Bridges Lab Protocols - User contributions [en]

Single fiber dissociation of intact rodent muscles

2016-05-09T20:39:50Z

Erinstephenson:

==Materials==
1. Dissection tools

2. Incubation medium (warm to 37 deg C)
*D-MEM (high glucose), containing:
#2% FBS
#5% PSG
#1mM Na Pyruvate

3. Dissociation medium (warm to 37 deg C)
*Incubation medium plus 4 mg/mL type-2 Collagenase

4. 35 mm cell culture dishes (or 6-well plates)

5. Glass Pasteur pipettes (1 mm bore) & pipette bulb

6. Incubator (37 deg C, 5% CO2)

7. 5 mL tubes

==Protocol==
#Anesthetize & CD mouse
#Dissect out muscle/muscles of interest
#Place into culture dish containing 2 mL of incubation medium & allow to recover in incubator (15-30 min)
#Carefully remove the medium & replace with 4 mL of dissociation medium
#Incubate for 2 hr
#Prepare fresh culture dishes containing 2-4 mL incubation medium
#Using a Pasteur pipette, carefully remove the muscle & place in new culture dish. Take care not to transfer too much of the dissociation medium.
#Gently pipette the muscle up & down to mechanically separate the muscle fibers. After 10 passes, return the remaining muscle bundle to the dissociation medium & return to the incubator for 20 min.
#Repeat last two steps. You may need to repeat this 2-3 times before the connective tissue is digested enough to yield ~80-90% of the muscle as single fibers.
#Remove debris (tendons, blood vessels, nerves, undigested muscle) using fine forceps & allow fibers to recover in the incubator for 30 min (overnight if additional cleaning is not required).
##Optional additional cleaning step: Transfer fibers in incubation medium to 5 mL tubes & allow to settle via gravity sedimentation (approx. 20 min). Carefully remove medium & replace with fresh incubation medium. Repeat once more, before placing cleaned fibers into fresh culture dishes.
#Let muscle fibers recover overnight. They are now ready for use experimentally.

==References==
This protocol is a modified method of:
*Cho et al., (2016) FASEB J 30(2):674-87 http://www.fasebj.org/content/30/2/674.full
*Spangenburg et al. http://www.seahorsebio.com/resources/tech-writing/techbrief-intact-muscle-fiber.pdf

Single fiber dissociation of intact rodent muscles

2016-05-09T20:34:26Z

Erinstephenson: This protocol is for isolating single muscle fibers from intact mouse skeletal muscles. It is optimized for use with the Flexor Digitorum Brevis (superficial foot) muscle.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2015-06-12T15:38:52Z

Erinstephenson:

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

== Experimental protocol ==
Note: Mice do not need to be in a fasted state prior to this test.
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice. ***Note: Take care when anesthetizing mice the second time. They will go under VERY quickly, so watch the mouse closely***
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
*Note: The manufacturers protocol says to use 225 uL of reaction buffer A and 75 uL of reaction buffer B with 4 uL of sample, read at 560:670 nm. This uses more reagent than is necessary for the volume of serum/concentrations of NEFA that we would typically analyze.

#To wells of a clear 96 well plate, add 4uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 0.25 and 0.5 mEq/L standards. For the high standard, add 8 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 4 uL of serum samples. ***Note: You may need to add more/less serum, depending on the NEFA concentration of your samples. The assay is linear between 0.01-4.00 mEq/L***
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 560:660 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 560 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes. The wells will turn purple. If they do not, check whether the reagents are within the best before date.
#Allow plate to return to room temperature before re-reading at 560:660 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 670 nm wavelength from those obtained at the 560 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2015-03-10T15:47:34Z

Erinstephenson: /* NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) */

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

== Experimental protocol ==
Note: Mice do not need to be in a fasted state prior to this test.
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice. ***Note: Take care when anesthetizing mice the second time. They will go under VERY quickly, so watch the mouse closely***
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
*Note: This page has been updated from a previous version, according to an update on the manufacturers website. Our previous method utilized 2.5 uL serum/standards in 100 uL reaction buffer A and 50 uL reaction buffer B, read at 550:660 nm

#To wells of a clear 96 well plate, add 4uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 0.25 and 0.5 mEq/L standards. For the high standard, add 8 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 4 uL of serum samples. ***Note: You may need to add more/less serum, depending on the NEFA concentration of your samples. The assay is linear between 0.01-4.00 mEq/L***
#To each well, add 225 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 560:670 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 560 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 75 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes. The wells will turn purple. If they do not, check whether the reagents are within the best before date.
#Allow plate to return to room temperature before re-reading at 560:670 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 670 nm wavelength from those obtained at the 560 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2015-03-10T15:44:30Z

Erinstephenson: /* NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) */

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

== Experimental protocol ==
Note: Mice do not need to be in a fasted state prior to this test.
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice. ***Note: Take care when anesthetizing mice the second time. They will go under VERY quickly, so watch the mouse closely***
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
*Note: This page has been updated from a previous version, according to an update on the manufacturers website. Our previous method utilized 2.5 uL serum/standards in 100 uL reaction buffer A and 50 uL reaction buffer B, read at 550:660 nm

#To wells of a clear 96 well plate, add 4uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 0.25 and 0.5 mEq/L standards. For the high standard, add 8 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 4 uL of serum samples. ***Note: You may need to add more/less serum, depending on the NEFA concentration of your samples***
#To each well, add 225 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 560:670 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 560 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 75 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes. The wells will turn purple. If they do not, check whether the reagents are within the best before date.
#Allow plate to return to room temperature before re-reading at 560:670 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 670 nm wavelength from those obtained at the 560 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2015-03-10T15:44:05Z

Erinstephenson: /* NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) */

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

== Experimental protocol ==
Note: Mice do not need to be in a fasted state prior to this test.
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice. ***Note: Take care when anesthetizing mice the second time. They will go under VERY quickly, so watch the mouse closely***
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
***Note: This page has been updated from a previous version, according to an update on the manufacturers website. Our previous method utilized 2.5 uL serum/standards in 100 uL reaction buffer A and 50 uL reaction buffer B, read at 550:660 nm***
#To wells of a clear 96 well plate, add 4uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 0.25 and 0.5 mEq/L standards. For the high standard, add 8 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 4 uL of serum samples. ***Note: You may need to add more/less serum, depending on the NEFA concentration of your samples***
#To each well, add 225 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 560:670 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 560 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 75 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes. The wells will turn purple. If they do not, check whether the reagents are within the best before date.
#Allow plate to return to room temperature before re-reading at 560:670 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 670 nm wavelength from those obtained at the 560 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2015-03-10T15:21:30Z

Erinstephenson: /* Experimental protocol */

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

== Experimental protocol ==
Note: Mice do not need to be in a fasted state prior to this test.
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice. ***Note: Take care when anesthetizing mice the second time. They will go under VERY quickly, so watch the mouse closely***
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 550:560 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 550 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before re-reading at 550:560 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 560 nm wavelength from those obtained at the 550 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Extraction of DNA from TRIZOL preparations

2015-02-19T20:49:55Z

Erinstephenson:

This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed. It is based on the online protocol by Shirley Zhu of the Chang Lab (http://changlab.stanford.edu/DNAextractionfromTRIZOL_Organicphase_update).

== '''Initial sample preparation''' ==
#Perform TRIZOL extraction as you normally would for RNA extraction.
#After removing the RNA-containing aqueous (upper) phase, re-centrifuge tubes (now containing only the interphase and organic (lower) phase) at 12,000 G for 5 min at 4 deg C.
#Remove any remaining upper phase, taking care to remove everything without damaging the interphase. Samples can now be stored at 4 deg C for days to a couple of weeks.

== '''DNA extraction''' ==
=== You will need: ===
==== 1. Back Extraction Buffer====
Containing 4 M Guanidine Thiocyanate (FW 118.16), 50 mM Sodium Citrate NaCi (FW 294.1) and 1 M Tris base (FW 121.14), prepared in ddH2O and sterile filtered.
For 25 mL, combine 11.82 g Guanidine Thiocyanate, 0.37 g Sodium Citrate NaCi and 3.03 g Tris base. Be careful to add small volumes of water, gradually, as the reagents will come up to the required volume VERY quickly.
==== 2. Isopropanol====
==== 3. 70% Ethanol====
==== 4. Qiagen Elution Buffer (or similar TE buffer)====

=== Extraction steps ===
#Add 500 uL of Back Extraction Buffer (BEB) for every 1 mL of TRIZOL used in the initial extraction. Place on shaker for 10 min. Always mix by inversion or use the shaker. NEVER vortex samples, as it will destroy the DNA.
#Centrifuge tubes at 12,000 G for 30 min at room temperature.
##Note: After this step is complete, set the centrifuge to cool to 4 deg C.
#Transfer aqueous (upper) phase to a clean tube and add 400 uL of ice cold isopropanol (to precipitate the DNA). Mix by inversion. Incubate at room temperature for 5 min.
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove supernatant (it is very unlikely that the DNA pellet will be visible). Add 500 uL of 70% ethanol and mix by inversion (to wash the pellet- this step removes the salt that co-precipitates with the DNA).
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove all of the supernatant (once again taking care not to disturb what is probably an invisible DNA pellet).
#Dissolve DNA pellet in 50 uL of Qiagen elution buffer. Samples can now be stored at 4 deg C.
##Note: Additional washing steps (using phenol, chloroform and isoamylalcohol) should be performed if a more pure pellet is required, however, this preparation should be sufficient for running qPCR/copy number experiments.

Extraction of DNA from TRIZOL preparations

2015-02-19T20:48:57Z

Erinstephenson: /* Extraction steps */

This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed. It is based on the online protocol by Shirley Zhu of the Chang Lab (http://changlab.stanford.edu/DNAextractionfromTRIZOL_Organicphase_update).

== '''Initial sample preparation''' ==
#Perform TRIZOL extraction as you normally would for RNA extraction.
#After removing the RNA-containing aqueous (upper) phase, re-centrifuge tubes (now containing only the interphase and organic (lower) phase) at 12,000 G for 5 min at 4 deg C.
#Remove any remaining upper phase, taking care to remove everything without damaging the interphase. Samples can now be stored at 4 deg C for days to a couple of weeks.

== '''DNA extraction''' ==
=== You will need: ===
====Back Extraction Buffer====
Containing 4 M Guanidine Thiocyanate (FW 118.16), 50 mM Sodium Citrate NaCi (FW 294.1) and 1 M Tris base (FW 121.14), prepared in ddH2O and sterile filtered.
For 25 mL, combine 11.82 g Guanidine Thiocyanate, 0.37 g Sodium Citrate NaCi and 3.03 g Tris base. Be careful to add small volumes of water, gradually, as the reagents will come up to the required volume VERY quickly.
====Isopropanol====
====70% Ethanol====
====Qiagen Elution Buffer (or similar TE buffer)====

=== Extraction steps ===
#Add 500 uL of Back Extraction Buffer (BEB) for every 1 mL of TRIZOL used in the initial extraction. Place on shaker for 10 min. Always mix by inversion or use the shaker. NEVER vortex samples, as it will destroy the DNA.
#Centrifuge tubes at 12,000 G for 30 min at room temperature.
##Note: After this step is complete, set the centrifuge to cool to 4 deg C.
#Transfer aqueous (upper) phase to a clean tube and add 400 uL of ice cold isopropanol (to precipitate the DNA). Mix by inversion. Incubate at room temperature for 5 min.
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove supernatant (it is very unlikely that the DNA pellet will be visible). Add 500 uL of 70% ethanol and mix by inversion (to wash the pellet- this step removes the salt that co-precipitates with the DNA).
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove all of the supernatant (once again taking care not to disturb what is probably an invisible DNA pellet).
#Dissolve DNA pellet in 50 uL of Qiagen elution buffer. Samples can now be stored at 4 deg C.
##Note: Additional washing steps (using phenol, chloroform and isoamylalcohol) should be performed if a more pure pellet is required, however, this preparation should be sufficient for running qPCR/copy number experiments.

Extraction of DNA from TRIZOL preparations

2015-02-19T20:46:30Z

Erinstephenson: This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed

This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed. It is based on the online protocol by Shirley Zhu of the Chang Lab (http://changlab.stanford.edu/DNAextractionfromTRIZOL_Organicphase_update).

== '''Initial sample preparation''' ==
#Perform TRIZOL extraction as you normally would for RNA extraction.
#After removing the RNA-containing aqueous (upper) phase, re-centrifuge tubes (now containing only the interphase and organic (lower) phase) at 12,000 G for 5 min at 4 deg C.
#Remove any remaining upper phase, taking care to remove everything without damaging the interphase. Samples can now be stored at 4 deg C for days to a couple of weeks.

== '''DNA extraction''' ==
=== You will need: ===
====Back Extraction Buffer====
Containing 4 M Guanidine Thiocyanate (FW 118.16), 50 mM Sodium Citrate NaCi (FW 294.1) and 1 M Tris base (FW 121.14), prepared in ddH2O and sterile filtered.
For 25 mL, combine 11.82 g Guanidine Thiocyanate, 0.37 g Sodium Citrate NaCi and 3.03 g Tris base. Be careful to add small volumes of water, gradually, as the reagents will come up to the required volume VERY quickly.
====Isopropanol====
====70% Ethanol====
====Qiagen Elution Buffer (or similar TE buffer)====

=== Extraction steps ===
#Add 500 uL of Back Extraction Buffer (BEB) for every 1 mL of TRIZOL used in the initial extraction. Place on shaker for 10 min.
#Centrifuge tubes at 12,000 G for 30 min at room temperature.
##Note: After this step is complete, set the centrifuge to cool to 4 deg C.
#Transfer aqueous (upper) phase to a clean tube and add 400 uL of ice cold isopropanol (to precipitate the DNA). Mix by inversion. Incubate at room temperature for 5 min.
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove supernatant (it is very unlikely that the DNA pellet will be visible). Add 500 uL of 70% ethanol and mix by inversion (to wash the pellet- this step removes the salt that co-precipitates with the DNA).
#Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
#Very carefully remove all of the supernatant (once again taking care not to disturb what is probably an invisible DNA pellet).
#Dissolve DNA pellet in 50 uL of Qiagen elution buffer. Samples can now be stored at 4 deg C.
##Note: Additional washing steps (using phenol, chloroform and isoamylalcohol) should be performed if a more pure pellet is required, however, this preparation should be sufficient for running qPCR/copy number experiments.

H&E Staining

2014-10-13T22:18:36Z

Erinstephenson: Created page with "== Rehydrate tissue sections == #After cutting sections of tissue onto glass slides, de-pariffinize the tissue sections in xylene for 3 min. Repeat for a total of 3x 3 minute ..."

== Rehydrate tissue sections ==
#After cutting sections of tissue onto glass slides, de-pariffinize the tissue sections in xylene for 3 min. Repeat for a total of 3x 3 minute washes using fresh xylene each time
#Rehydrate the tissue sections by immersing in 100% ethanol for 3 minutes. Repeat a second time using fresh 100% ethanol
#Immerse sections in 95% ethanol for 3 minutes. Repeat a second time using fresh 95% ethanol
#Immerse sections in 70% ethanol for 3 minutes
#Rinse sections 2 to 3 times in distilled water

== Stain tissue sections with haematoxylin ==
#Immerse sections in pre-prepared Mayer's haematoxylin for 5 minutes
#Rinse under running tap water (slides still in box) until water is clear. This will take about 5 minutes

== Stain tissue sections with eosin, dehydrate and coverslip ==
#Immerse sections in 1% eosin from 15 seconds to 1 minute
#Rinse 3-4 times under running tap water
#Immerse in 70% ethanol for 2 minutes
#Immerse in in 95% ethanol for 2 minutes. Repeat a second time using fresh 95% ethanol
#Immerse in in 100% ethanol for 2 minutes. Repeat twice more using fresh 100% ethanol
#Immerse in xylene for 2 minutes. Repeat twice more using fresh xylene. Check staining under the microscope
#Coverslip using Permount or other xylene-based mounting medium

Paraffin Embedding of Tissue Samples

2014-09-22T21:44:04Z

Erinstephenson:

== Paraffin embedding protocol ==
#Harvest tissue from animal, place into cassette and submerge in 10% neutral buffered formalin for 24-48 hrs (depending on thickness of sample).
#Move cassettes into 70% ethanol for 24 hrs. Samples can be left in 70% for long term storage, if required.
##Note that the subsequent dehydration steps involve many long incubation periods and therefore should be started first thing in the morning (unless you plan on being in the lab overnight!).
#Move cassettes into 75% ethanol for 30 minutes.
#Move cassettes into 95% ethanol for 75 minutes. Repeat this step a second time, using fresh 95% ethanol.
#Move cassettes into 100% ethanol for 60 minutes. Repeat this step twice more, using fresh 100% ethanol each time.
##Some protocols suggest that the first 100% ethanol wash can be left over night if time is an issue. However, it must be noted that extending the dehydration process can result in alterations to the morphology of your tissue.
##During these incubation steps, prepare beakers with 58-60 deg C paraffin (the melting point of paraffin is 58 C; many embedding procedures recommend that the paraffin to be 2 C above the melting point for best results).
#Move cassettes into xylenes for 30-60 minutes. Repeat this step a second time, using fresh xylenes.
#Move cassettes into 60 deg C paraffin for 60 minutes. Repeat this step twice more, using fresh paraffin each time.
##Some protocols suggest that the second paraffin incubation step can be extended over night. However, this may increase the risk of the tissue cracking during sectioning.
##During these incubation steps, turn on the paraffin wax machine (Link Building, Room 311).
#Immediately prior to embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with melted paraffin.
#Take your sample and orient it on top of the base wax as desired. Partially cover with more paraffin.
#Place pathology cassette on top of the mold and completely fill the mold.
#Place mold with sample on "Cold Side" of embedding station .
##The paraffin will solidify in 10-15 minutes.
#Remove embedded sample from the mold (should just slip out).
#Store at room temperature until ready to section.

How to design primer pairs for determining the presence of CrispR-cas induced mutations

2014-08-14T21:52:25Z

Erinstephenson: Created page with "#Go to http://www.ncbi.nlm.nih.gov/nuccore/ and find the sequence of interest. #Scroll down to the sequence and highlight/copy the nucleotides corresponding with your CrispR ..."

#Go to http://www.ncbi.nlm.nih.gov/nuccore/ and find the sequence of interest.
#Scroll down to the sequence and highlight/copy the nucleotides corresponding with your CrispR cut site.
#Go to http://www.ncbi.nlm.nih.gov/gene/ and find your gene. Paste your cut sequence into the ‘Find’ box. A new dialog box will appear. Click on the ‘sequence’ tab and select your sequence. The CrispR cut site will appear highlighted on the gene map.
#Zoom out to where the visible sites flanking your cut site are an appropriate size for primer design (~70-150 bp).
#From the ‘Tools’ tab, select ‘BLAST and primer search’, followed by ‘Primer BLAST- visible range’. A new tab will open. In this new tab, scroll down and click ‘Get Primers’. Primer BLAST will run automatically.
#When the search results appear, paste your cut sequence into the ‘Find’ box, just like in step 3. You will be able to see which primer pairs flank your cut sequence and which do not. Based on these options you should be able to select a primer pair that does not overlap with your cut sequence.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2014-07-24T18:18:59Z

Erinstephenson:

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

== Experimental protocol ==
Note: Mice do not need to be in a fasted state prior to this test.
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 550:560 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 550 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before re-reading at 550:560 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 560 nm wavelength from those obtained at the 550 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2014-07-24T18:17:50Z

Erinstephenson:

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

Note: Mice do not need to be in a fasted state prior to this test.

== Experimental protocol ==
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 550:560 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 550 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before re-reading at 550:560 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 560 nm wavelength from those obtained at the 550 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2014-07-24T18:15:34Z

Erinstephenson:

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

Note: Mice do not need to be in a fasted state prior to this test.

== Experimental protocol ==
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 550:560 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 550 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before re-reading at 550:560 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 560 nm wavelength from those obtained at the 550 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present before lipolysis is stimulated.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after lipolysis has been stimulated and the triglycerides present have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2014-07-24T18:13:09Z

Erinstephenson:

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

Note: Mice do not need to be in a fasted state prior to this test.

== Experimental protocol ==
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 550:560 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 550 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before re-reading at 550:560 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 560 nm wavelength from those obtained at the 550 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after all the triglycerides have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2014-07-24T18:12:03Z

Erinstephenson:

== Assessing Isoproterenol-Stimulated Whole-Body Lipolysis ''in vivo'' ==
Background
#Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

Note: Mice do not need to be in a fasted state prior to this test.

Experimental protocol
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit)
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 550:560 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 550 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before re-reading at 550:560 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 560 nm wavelength from those obtained at the 550 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit)
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after all the triglycerides have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2014-07-24T17:28:35Z

Erinstephenson:

== Assessing Isoproterenol-Stimulated Whole-Body Lipolysis ''in vivo'' ==

Note: Mice do not need to be in a fasted state prior to this test.

Experimental protocol
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit)
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 550:560 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 550 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before re-reading at 550:560 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 560 nm wavelength from those obtained at the 550 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit)
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To remaining wells, add 3 uL of serum samples.
#To each well, add 80 uL of glycerol reagent. Incubate plate at 37 deg C for 30 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after all the triglycerides have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2014-07-24T14:48:21Z

Erinstephenson: Created page with "== Assessing Isoproterenol-Stimulated Whole-Body Lipolysis ''in vivo'' == ##Note: Mice do not need to be in a fasted state prior to this test. #Briefly anesthetize mice with ..."

== Assessing Isoproterenol-Stimulated Whole-Body Lipolysis ''in vivo'' ==

##Note: Mice do not need to be in a fasted state prior to this test.
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

NEFA determination from serum
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
#To each well, add 100 uL of reaction buffer A

Paraffin Embedding of Tissue Samples

2014-07-22T18:44:44Z

Erinstephenson:

== Protocol 1 ==
#After harvesting tissue from animal, place into pathology cassette and fix in 4% formaldehyde, 4% paraformaldehyde or 4% formalin O/N
#After fixation, begin dehydrating the tissue in 70% Ethanol
#On the day of planned embedding, wash sample with 100% Ethanol 3 times for 30 minutes.
##While washing in 100% Ethanol, start the paraffin wax machine (Link Building, Room 311)
#After 100% Ethanol washes, Wash 3 times in Xylenes
#Wash samples in a beaker filled with wax at 58-60C, 3 times for 15 mins
#Immediately before embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with wax
#Take your sample and orient it on top of the base wax as desired
#Place pathology cassette on top of the mold and fill with wax covering the mold
#Place mold with sample on "Cold Side" of embedding station
##Wax will solidify in 10-15 minutes
#Remove wax embedded sample from the mold (should just slip out)
#Store at room temperature until prepped to section

== Paraffin embedding protocol 2 ==
#Harvest tissue from animal, place into cassette and submerge in 10% neutral buffered formalin for 24-48 hrs (depending on thickness of sample).
#Move cassettes into 70% ethanol for 24 hrs. Samples can be left in 70% for long term storage, if required.
##Note that the subsequent dehydration steps involve many long incubation periods and therefore should be started first thing in the morning (unless you plan on being in the lab overnight!).
#Move cassettes into 75% ethanol for 30 minutes.
#Move cassettes into 95% ethanol for 75 minutes. Repeat this step a second time, using fresh 95% ethanol.
#Move cassettes into 100% ethanol for 60 minutes. Repeat this step twice more, using fresh 100% ethanol each time.
##Some protocols suggest that the first 100% ethanol wash can be left over night if time is an issue. However, it must be noted that extending the dehydration process can result in alterations to the morphology of your tissue.
##During these incubation steps, prepare beakers with 58-60 deg C paraffin (the melting point of paraffin is 58 C; many embedding procedures recommend that the paraffin to be 2 C above the melting point for best results).
#Move cassettes into xylenes for 30-60 minutes. Repeat this step a second time, using fresh xylenes.
#Move cassettes into 60 deg C paraffin for 60 minutes. Repeat this step twice more, using fresh paraffin each time.
##Some protocols suggest that the second paraffin incubation step can be extended over night. However, this may increase the risk of the tissue cracking during sectioning.
##During these incubation steps, turn on the paraffin wax machine (Link Building, Room 311).
#Immediately prior to embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with melted paraffin.
#Take your sample and orient it on top of the base wax as desired. Partially cover with more paraffin.
#Place pathology cassette on top of the mold and completely fill the mold.
#Place mold with sample on "Cold Side" of embedding station .
##The paraffin will solidify in 10-15 minutes.
#Remove embedded sample from the mold (should just slip out).
#Store at room temperature until ready to section.

Protocol Edited from: FFPE SOP - MKM and SR for the Williams Lab

1. Collect tissue
a. Immediately place in labeled cassette in 10% buffered formalin
b. Place in labeled cassette on dry ice prior to storage at -80C

2. Fix in 10% buffered formalin
a. NOTE: for 3 mm mouse brain sections NEVER exceed 24H in formalin

3. Tissue processing
a. 70% EtOH (30 min) → tissues can be kept in 70% Ethanol for long-term storage
b. 95% EtOH (30 min)
c. 100% EtOH (30-60 min)
d. Xylene (30 min under a fume hood)
e. Xylene (30 min under a fume hood)
f. Rinse tissues in Paraffin (melting temp of wax, 56 oC for paraplast X-tra)
g. Paraffin (melting temp of wax, 56 oC for paraplast X-tra)

*NOTE: Steps 3a to 3e can be done in bulk using plastic histology containers placed on a shaker. In steps 3f to 3g transfer the tissue to a 1.5 ml labeled eppendorf tube and add melted Paraffin → place in a 56 oC heated water bath and floating tube rack.

4. Embedding
a. Spray the stainless steel cassette with ‘base mold release agent’ prior to adding heated wax.
b. Pour a small amount (1/3 of the volume) of heated wax into a pre-heated stainless steel cassette and arrange tissue pressing it flat against the bottom with a pre-heated instrument.
c. Place the back side of the plastic labeled cassette on top of the stainless steel cassette and fill until wax just covers mesh.
d. Chill until paraffin is completely solidified and then remove stainless steel cassette.

Sectioning
1) Section 10-15 micron sections from cold blocks of paraffin imbedded tissue with cold blade (pre-chilled in 4 oC).
2) Pretreatment of paraffin tissue sections with either of the following
a. Leave slides at room temperature for 60 minutes; or
b. Heat in dry oven at 55°C for 20 minutes
Note 1: for adipose, leaving at room temperature prevents disruption of the tissue
Note 2: For RNA and antigens option (b) is recommended. Option (a) is for highly heat sensitive molecules.
3) Immerse slide in xylene for 2 min. (fat) or 10 min. (liver).
4) Immerse slide in 100% ethanol for 2 minutes.
5) Immerse slide in 95% ethanol for 1 minute.
6) Immerse slide in 70% ethanol for 1 minute.
7) Immerse slide in 50% ethanol for 1 minute.
8) Immerse slide in 1X PBS for 2 minutes.
9) Air dry slides for 10-20 min

Protocol Edited from: several online IHC protocols

H&E Staining

Hematoxylin staining
Incubate slides in Mayer’s Hematoxylin for 20-30 min (fat) or 10 min (liver)
Rinse in warm H2O (note ‘blueing’ of nuclei)

Eosin staining
Working solution:
• 25 mL of 1% Eosin (dissolved in 2 parts of ddH2O and 8 parts of 95% Ethanol)
• 75 mL of 80% Ethanol
• 0.5 mL Glacial Acetic Acid

Stain sections for 10 min. in 0.25% Eosin solution
Rinse slides in ddH2O

Dehydrate slides in
1) Immerse slide in 70% ethanol (4 dips)
2) Immerse slide in 95% ethanol (2 dips).
3) Immerse slide in 100% ethanol (2 dips).
4) Immerse slide in xylene for (6 dips)

Mounting
Apply a drop of mounting medium over the section
Place the cover slip over in an angle to avoid bubbles
Store slides at room temperature for 2 h prior to analysis

Paraffin Embedding of Tissue Samples

2014-07-22T18:24:56Z

Erinstephenson:

== Protocol 1 ==
#After harvesting tissue from animal, place into pathology cassette and fix in 4% formaldehyde, 4% paraformaldehyde or 4% formalin O/N
#After fixation, begin dehydrating the tissue in 70% Ethanol
#On the day of planned embedding, wash sample with 100% Ethanol 3 times for 30 minutes.
##While washing in 100% Ethanol, start the paraffin wax machine (Link Building, Room 311)
#After 100% Ethanol washes, Wash 3 times in Xylenes
#Wash samples in a beaker filled with wax at 58-60C, 3 times for 15 mins
#Immediately before embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with wax
#Take your sample and orient it on top of the base wax as desired
#Place pathology cassette on top of the mold and fill with wax covering the mold
#Place mold with sample on "Cold Side" of embedding station
##Wax will solidify in 10-15 minutes
#Remove wax embedded sample from the mold (should just slip out)
#Store at room temperature until prepped to section

== Paraffin embedding protocol 2 ==
#Harvest tissue from animal, place into cassette and submerge in 10% neutral buffered formalin for 24-48 hrs (depending on thickness of sample).
#Move cassettes into 70% ethanol for 24 hrs. Samples can be left in 70% for long term storage. Note that the subsequent dehydration steps involve long incubation periods and therefore should be started first thing in the morning (unless you plan on being in the lab overnight!).
#Move cassettes into 75% ethanol for 30 minutes.
#Move cassettes into 95% ethanol for 75 minutes. Repeat this step a second time, using fresh 95% ethanol.
#Move cassettes into 100% ethanol for 60 minutes. Repeat this step twice more, using fresh 100% ethanol each time.
##During these incubation steps, prepare beakers with 60 deg C paraffin.
#Move cassettes into xylenes for 30-60 minutes. Repeat this step a second time, using fresh xylenes.
#Move cassettes into 60 deg C paraffin for 60 minutes. Repeat this step twice more, using fresh paraffin each time. The second paraffin incubation step can be extended over night, if necessary.
##During these incubation steps, turn on the paraffin wax machine (Link Building, Room 311).
#Immediately prior to embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with wax.
#Take your sample and orient it on top of the base wax as desired. Partially cover with more paraffin.
#Place pathology cassette on top of the mold and completely fill the mold.
#Place mold with sample on "Cold Side" of embedding station .
##Wax will solidify in 10-15 minutes.
#Remove wax embedded sample from the mold (should just slip out).
#Store at room temperature until ready to section.

Protocol Edited from: FFPE SOP - MKM and SR for the Williams Lab

1. Collect tissue
a. Immediately place in labeled cassette in 10% buffered formalin
b. Place in labeled cassette on dry ice prior to storage at -80C

2. Fix in 10% buffered formalin
a. NOTE: for 3 mm mouse brain sections NEVER exceed 24H in formalin

3. Tissue processing
a. 70% EtOH (30 min) → tissues can be kept in 70% Ethanol for long-term storage
b. 95% EtOH (30 min)
c. 100% EtOH (30-60 min)
d. Xylene (30 min under a fume hood)
e. Xylene (30 min under a fume hood)
f. Rinse tissues in Paraffin (melting temp of wax, 56 oC for paraplast X-tra)
g. Paraffin (melting temp of wax, 56 oC for paraplast X-tra)

*NOTE: Steps 3a to 3e can be done in bulk using plastic histology containers placed on a shaker. In steps 3f to 3g transfer the tissue to a 1.5 ml labeled eppendorf tube and add melted Paraffin → place in a 56 oC heated water bath and floating tube rack.

4. Embedding
a. Spray the stainless steel cassette with ‘base mold release agent’ prior to adding heated wax.
b. Pour a small amount (1/3 of the volume) of heated wax into a pre-heated stainless steel cassette and arrange tissue pressing it flat against the bottom with a pre-heated instrument.
c. Place the back side of the plastic labeled cassette on top of the stainless steel cassette and fill until wax just covers mesh.
d. Chill until paraffin is completely solidified and then remove stainless steel cassette.

Sectioning
1) Section 10-15 micron sections from cold blocks of paraffin imbedded tissue with cold blade (pre-chilled in 4 oC).
2) Pretreatment of paraffin tissue sections with either of the following
a. Leave slides at room temperature for 60 minutes; or
b. Heat in dry oven at 55°C for 20 minutes
Note 1: for adipose, leaving at room temperature prevents disruption of the tissue
Note 2: For RNA and antigens option (b) is recommended. Option (a) is for highly heat sensitive molecules.
3) Immerse slide in xylene for 2 min. (fat) or 10 min. (liver).
4) Immerse slide in 100% ethanol for 2 minutes.
5) Immerse slide in 95% ethanol for 1 minute.
6) Immerse slide in 70% ethanol for 1 minute.
7) Immerse slide in 50% ethanol for 1 minute.
8) Immerse slide in 1X PBS for 2 minutes.
9) Air dry slides for 10-20 min

Protocol Edited from: several online IHC protocols

H&E Staining

Hematoxylin staining
Incubate slides in Mayer’s Hematoxylin for 20-30 min (fat) or 10 min (liver)
Rinse in warm H2O (note ‘blueing’ of nuclei)

Eosin staining
Working solution:
• 25 mL of 1% Eosin (dissolved in 2 parts of ddH2O and 8 parts of 95% Ethanol)
• 75 mL of 80% Ethanol
• 0.5 mL Glacial Acetic Acid

Stain sections for 10 min. in 0.25% Eosin solution
Rinse slides in ddH2O

Dehydrate slides in
1) Immerse slide in 70% ethanol (4 dips)
2) Immerse slide in 95% ethanol (2 dips).
3) Immerse slide in 100% ethanol (2 dips).
4) Immerse slide in xylene for (6 dips)

Mounting
Apply a drop of mounting medium over the section
Place the cover slip over in an angle to avoid bubbles
Store slides at room temperature for 2 h prior to analysis

Rapamycin Injections

2014-02-17T17:35:07Z

Erinstephenson:

==Materials==
*Tween 80 - Sigma P1754
*PEG 400 Liquid - Sigma P3265

==Preparation==
*Weigh out 2.08g of each Tween 80 and PEG into a 50 mL falcon tube. Bring up to 40 mL with water and filter by steriflip. This is 5.2% PEG/Tween
*Dissolve rapamycin at 20 mg/mL (0.0219 M) in ethanol (prepare as a 1 mL aliquot and store at -20 C).
*Add 25 uL of 20 mg/mL RAPA in 5 mL Tween 80/PEG diluent to get a 0.1 mg/mL concentration (109 uM; use this for 1 week).
*IP inject 10 uL/g body weight (1 mg/kg)

==Alternate Rapamycin Injection Regimen==
* Phenotypes seem to be different with rapamycin as treated by Lamming et al.
* This protocol is from Lamming et al http://dx.doi.org/10.1126/science.1215135
<blockquote>
rapamycin treatment was performed by injecting 8 to 10 week old mice intraperitoneally
once daily with either 1 mg/kg rapamycin suspended in 0.9% NaCl and 2% ethanol at a
concentration of 0.5 mg/mL (547 μM), or vehicle only for 14-28 days.
</blockquote>

Rapamycin Injections

2014-02-17T17:24:01Z

Erinstephenson:

==Materials==
*Tween 80 - Sigma P1754
*PEG 400 Liquid - Sigma P3265

==Preparation==
*Weight out 2.08g of each Tween 80 and PEG into a 50 mL falcon tube. Bring up to 40 mL with water and filter by steriflip. This is 5.2% PEG/Tween
*Dissolve rapamycin at 20 mg/mL in water.
*Add 25 uL of 20 mg/mL RAPA in 5 mL Tween 80/PEG diluent to get a 0.1 mg/mL concentration (use this for 1 week).
*Inject 10 uL/g body weight

==Alternate Rapamycin Injection Regimen==
* Phenotypes seem to be different with rapamycin as treated by Lamming et al.
* This protocol is from Lamming et al http://dx.doi.org/10.1126/science.1215135
<blockquote>
rapamycin treatment was performed by injecting 8 to 10 week old mice intraperitoneally
once daily with either 1 mg/kg rapamycin suspended in 0.9% NaCl and 2% ethanol at a
concentration of 0.5 mg/mL (547 μM), or vehicle only for 14-28 days.
</blockquote>