Bridges Lab Protocols - User contributions [en]

H&E Staining of Mouse Tissue

2015-06-09T14:51:58Z

Aragaus1: /* Protocol */

==Materials==
*xylene/Citrisolv
*100% Ethanol
*70% Ethanol
*H2O
*Mayer's Hematoxylin
*1% Eosin Y (in 95% ethanol)
*95% Ethanol

==Protocol==
*Removing Wax and Rehydrating
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
# Deparaffinize and rehydrate sections
## Immerse for 5 min with agitation in xylene/Citrisolv
## Immerse for 5 min with agitation in 100% ethanol
## Immerse for 5 min with agitation in 70% ethanol
## Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min
*H&E staining
#Dip slide into Mayer's Hematoxylin and agitate for 30 sec
#Rinse slide in H2O for 1 minute
##depending on the intensity desired, repeat this step if necessary
#Dip slide in 1% eosin Y fo 10-30 sec with agitation
##For adipose tissue, 20 seconds allows for proper staining.
*Dehydration
#Place slides in 95% Ethanol two times for 30 seconds each.
#Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
#Wash twice with fresh xylene/Citrisolv for 30 seconds.
#Let slide dry.
#Place 1-2 drops of mounting medium on slide and place coverslip over
#Seal coverslip with sealant(nailpolish)

==Notes==
*Be sure to shake Eosin prior to placing slide in for staining because it will settle.
*The staining will get more intense after time, be sure not to oversaturate section
*Especially during the summer months, be sure to not let the slides set out overnight/extended period of time.

*Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986

H&E Staining of Mouse Tissue

2015-06-09T14:51:30Z

Aragaus1: /* Materials */

==Materials==
*xylene/Citrisolv
*100% Ethanol
*70% Ethanol
*H2O
*Mayer's Hematoxylin
*1% Eosin Y (in 95% ethanol)
*95% Ethanol

==Protocol==
*Removing Wax and Rehydrating
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
# Deparaffinize and rehydrate sections
## Immerse for 5 min with agitation in xylene
## Immerse for 5 min with agitation in 100% ethanol
## Immerse for 5 min with agitation in 70% ethanol
## Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min
*H&E staining
#Dip slide into Mayer's Hematoxylin and agitate for 30 sec
#Rinse slide in H2O for 1 minute
##depending on the intensity desired, repeat this step if necessary
#Dip slide in 1% eosin Y fo 10-30 sec with agitation
##For adipose tissue, 20 seconds allows for proper staining.
*Dehydration
#Place slides in 95% Ethanol two times for 30 seconds each.
#Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
#Wash twice with fresh xylene for 30 seconds.
#Let slide dry.
#Place 1-2 drops of mounting medium on slide and place coverslip over
#Seal coverslip with sealant(nailpolish)

==Notes==
*Be sure to shake Eosin prior to placing slide in for staining because it will settle.
*The staining will get more intense after time, be sure not to oversaturate section
*Especially during the summer months, be sure to not let the slides set out overnight/extended period of time.

*Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986

H&E Staining of Mouse Tissue

2015-06-09T14:51:04Z

Aragaus1: /* Notes */

==Materials==
*xylene
*100% Ethanol
*70% Ethanol
*H2O
*Mayer's Hematoxylin
*1% Eosin Y (in 95% ethanol)
*95% Ethanol

==Protocol==
*Removing Wax and Rehydrating
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
# Deparaffinize and rehydrate sections
## Immerse for 5 min with agitation in xylene
## Immerse for 5 min with agitation in 100% ethanol
## Immerse for 5 min with agitation in 70% ethanol
## Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min
*H&E staining
#Dip slide into Mayer's Hematoxylin and agitate for 30 sec
#Rinse slide in H2O for 1 minute
##depending on the intensity desired, repeat this step if necessary
#Dip slide in 1% eosin Y fo 10-30 sec with agitation
##For adipose tissue, 20 seconds allows for proper staining.
*Dehydration
#Place slides in 95% Ethanol two times for 30 seconds each.
#Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
#Wash twice with fresh xylene for 30 seconds.
#Let slide dry.
#Place 1-2 drops of mounting medium on slide and place coverslip over
#Seal coverslip with sealant(nailpolish)

==Notes==
*Be sure to shake Eosin prior to placing slide in for staining because it will settle.
*The staining will get more intense after time, be sure not to oversaturate section
*Especially during the summer months, be sure to not let the slides set out overnight/extended period of time.

*Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986

H&E Staining of Mouse Tissue

2015-06-08T16:36:43Z

Aragaus1: /* Protocol */

==Materials==
*xylene
*100% Ethanol
*70% Ethanol
*H2O
*Mayer's Hematoxylin
*1% Eosin Y (in 95% ethanol)
*95% Ethanol

==Protocol==
*Removing Wax and Rehydrating
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
# Deparaffinize and rehydrate sections
## Immerse for 5 min with agitation in xylene
## Immerse for 5 min with agitation in 100% ethanol
## Immerse for 5 min with agitation in 70% ethanol
## Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min
*H&E staining
#Dip slide into Mayer's Hematoxylin and agitate for 30 sec
#Rinse slide in H2O for 1 minute
##depending on the intensity desired, repeat this step if necessary
#Dip slide in 1% eosin Y fo 10-30 sec with agitation
##For adipose tissue, 20 seconds allows for proper staining.
*Dehydration
#Place slides in 95% Ethanol two times for 30 seconds each.
#Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
#Wash twice with fresh xylene for 30 seconds.
#Let slide dry.
#Place 1-2 drops of mounting medium on slide and place coverslip over
#Seal coverslip with sealant(nailpolish)

==Notes==
*Be sure to shake Eosin prior to placing slide in for staining because it will settle.
*The staining will get more intense after time, be sure not to oversaturate section

*Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986

H&E Staining of Mouse Tissue

2015-06-08T16:36:02Z

Aragaus1: /* Materials */

==Materials==
*xylene
*100% Ethanol
*70% Ethanol
*H2O
*Mayer's Hematoxylin
*1% Eosin Y (in 95% ethanol)
*95% Ethanol

==Protocol==
*Removing Wax and Rehydrating
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
# Deparaffinize and rehydrate sections
## Immerse for 5 min with agitation in xylene
## Immerse for 5 min with agitation in 100% ethanol
## Immerse for 5 min with agitation in 70% ethanol
## Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min
*H&E staining
#Dip slide into Mayer's Hematoxylin and agitate for 30 sec
#Rinse slide in H2O for 1 minute
##depending on the intensity desired, repeat this step if necessary
#Dip slide in 1% eosin Y fo 10-30 sec with agitation
*Dehydration
#Place slides in 95% Ethanol two times for 30 seconds each.
#Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
#Wash twice with fresh xylene for 30 seconds.
#Let slide dry.
#Place 1-2 drops of mounting medium on slide and place coverslip over
#Seal coverslip with sealant(nailpolish)

==Notes==
*Be sure to shake Eosin prior to placing slide in for staining because it will settle.
*The staining will get more intense after time, be sure not to oversaturate section

*Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986

Maternal Particulate Exposure Breeding

2015-04-02T17:00:57Z

Aragaus1: Created page with "==Ordering== *Order C57BL6 mice at 42 days of age from jaxmice.org *Once the mice arrive, place mice on breeder's chow for seven days *At 49 days of age, mate female and male ..."

==Ordering==
*Order C57BL6 mice at 42 days of age from jaxmice.org
*Once the mice arrive, place mice on breeder's chow for seven days
*At 49 days of age, mate female and male mice.
==Exposure==
*On day 10 and 17 of gestation expose the mice to particulate or control (saline/cabosil).
**Should be close to days 59 and 66 days of age.
==Weaning==
*Keep the mother's on breeder's chow through weaning.

Main Page

2015-04-02T16:28:04Z

Aragaus1:

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
*[[Maternal Particulate Exposure Breeding]]

==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]
*[[H&E Staining of Mouse Tissue]]
**[[Adipocyte Cell Counting with ImageJ]]
*[[Perfusion of Mouse]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Main Page

2015-04-02T16:27:40Z

Aragaus1: /* Mouse/Fly Protocols */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
*Maternal Particulate Exposure Breeding

==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]
*[[H&E Staining of Mouse Tissue]]
**[[Adipocyte Cell Counting with ImageJ]]
*[[Perfusion of Mouse]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Main Page

2015-04-02T16:27:27Z

Aragaus1: /* Mouse/Fly Protocols */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
*Maternal Particulate Exposure Breeding Protocol

==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]
*[[H&E Staining of Mouse Tissue]]
**[[Adipocyte Cell Counting with ImageJ]]
*[[Perfusion of Mouse]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Adipocyte Cell Counting with ImageJ

2015-03-31T18:40:34Z

Aragaus1:

==ImageJ Download==
*Download ImageJ from http://imagej.nih.gov/ij/download.html
**To to open ImageJ, right click on software in applications folder
***Since the program is downloaded from the internet it will not open up automatically and you must go through applications folder.
==Adipocyte Counting Macro==
*Download adipocyte counting tool from http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Adipocytes_Tool
*Open ImageJ
*Click on Plugins
**Macros
***Install
****Select "MRI_Adipocyte_Tools" from downloads to install.
==Adipocyte Cell Counting==
*With macro installed, click on segmentation: "s"
**Set boundaries for sizing of cell
***Use 40-40,000 for size typically.
**Change thresholding method to Default.
*Open image of interest.
*Click on segmentation "s"
*Select freehanding tool to outline any cells that were not counted
**To add freehanded cell to ROI manager, click command + T
*Once all cells have been outlined, and misnumbered/outlined cells have been removed, click on measure
**Copy and save measurements to Excel spreadsheet
===To save image of outlined cells===
*Make sure all cells are outlined.
*Click on "Flatten"
*Save flattened image to folder.

Adipocyte Cell Counting with ImageJ

2015-03-31T18:34:30Z

Aragaus1: Created page with "==ImageJ Download== *Download ImageJ from http://imagej.nih.gov/ij/download.html **To to open ImageJ, right click on software in applications folder ***Since the program is do..."

Main Page

2015-03-31T18:21:51Z

Aragaus1: /* Tissue Preparation */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]
*[[H&E Staining of Mouse Tissue]]
**[[Adipocyte Cell Counting with ImageJ]]
*[[Perfusion of Mouse]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Perfusion of Mouse

2015-03-27T19:14:19Z

Aragaus1:

==Materials==
*0.9 % Saline solution
*10% formalin solution
*Avertin
*Isofluorane
*30 gauge needle
*1 mL syringe
*Peristaltic pump
*Collecting dish
*Surgical stage/platform
**Make sure this is level with the edge of the collecting dish to allow for the need to rest during the procedure
===Surgical tools:===
*Dissecting scissors
*Forceps
*Clamp scissors
*Standard scissors

===Avertin Preparation===
*Taken from: http://web.jhu.edu/animalcare/rdf/avertin.html
*Stock Solution (1.6 g/ml)
**Mix 25 g avertin and 15.5 ml tert-amyl alcohol at room temperture for ~12 hours in a dark bottle.
**Can store stock solutions at room temperature for one year.
*Working solution (20 mg/ml)
**Mix 0.5 ml avertin stock solution and 39.5 ml 0.9% saline in dark/foil covered container.
**Filter solution through 0.2 micron filter into a dark/foiled covered container.
**Store solution at 4 degree C.
**Replace working solution each month.
*Notes:
**Avertin is light sensitive.
**Degradation products are lethal.
**Always store at 4 degree C
**Do NOT use a solution that is yellow or contains a precipitate.
**Avertin is lipid soluble and may require a larger dose in an obese animal.

==Perfusion Pump Set Up==
*Place needle on end of tubing
*Insert one tube into 0.9% saline solution, and one tube into 10% formalin solution
''Image 1: Set up of the two tube system from 0.9% saline solution and 10% formalin solution.''
[[File:perfusionbottle.png]]
*Turn pump on to a flow rate of 0.72 ml/min
*Have stopcock turned to allow for flow of saline
''Image 2: Stopcock set up to allow for 0.9% saline and 10% formalin solution to flow through to main line.''
[[File:perfusionstopcock.png]]
*Fill line with 0.9% saline until you have a steady stream of 0.9% saline from the end of the needle.
*Turn off peristaltic pump and turn stopcock to allow for formalin solution to flow
*Turn on peristaltic pump and pump 10% formalin solution until flow fluid is before the stopcock
**Flow has not entered the main line
*Turn off peristaltic pump and turn stopcock to allow for saline to flow
*Turn on pump and pump saline through the main line at stated flow rate
''Image 3: Overview of the entire perfusion setup (excluding the procedural platform in the collecting dish)''
[[File:perfusionsetup.png]]

==Animal Procedure==
*Place mouse in isofluorane chamber and lightly anesthetize the animal.
*Remove mouse from chamber
*For mouse anesthesia, administer 0.8 ml/20 g (of mouse body weight) Avertin through intraperitoneal injection with 30 ½ gauge needle.
*Wait for 3 minutes or until the mouse no longer responds to painful stimuli, such as paw pinch before proceeding.
*Lay the mouse on its back.
*Using tweezers and operating/dissecting scissors open up the skin and expose the chest cavity.
*Cut open the diaphragm using standard scissors.
**Be careful to not pierce the heart.
*Using clamp scissors, grab at the base of the sternum, cut through the ribcage and lift to expose the heart.
*Transfer the mouse to the procedural stage/platform.
*Insert the 30 gauge needle (from the tubing with saline/10% formalin solution) into the apex of the left ventricle, being careful to keep the tip of the needle in the lumen of the ventricle.
*Immediately after inserting the needle into the left ventricle, cut the right ventricle using standard scissors.
**This allows for the blood to flow out of the mouse and drain into collecting dish.
*Perfuse with saline solution for 10 minutes.
*After 10 minutes, switch the stopcock to allow for flow of formalin solution, at same flow rate as saline.
*Perfuse for 10-12 minutes with 10% formalin solution.
*With the pump still on, remove needle from left ventricle.
**Mouse is now fixed for tissue collection.
*When collecting tissues, keep tissues in 10% formalin solution for ~24 hours at 4 degree C.
*Transfer tissues to PBS after for long-term storage at 4 degree C.

File:Perfusionsetup.png

2015-03-27T17:35:40Z

Aragaus1:

Perfusion of Mouse

2015-03-27T17:35:21Z

Aragaus1:

==Materials==
*0.9 % Saline solution
*10% formalin solution
*Avertin
*Isofluorane
*30 ½ gauge needle
*1 mL syringe
*Peristaltic pump
*Collecting dish
*Surgical stage/platform
**Make sure this is level with the edge of the collecting dish to allow for the need to rest during the procedure
===Surgical tools:===
*Dissecting scissors
*Forceps
*Clamp scissors
*Standard scissors

===Avertin Preparation===
*Taken from: http://web.jhu.edu/animalcare/rdf/avertin.html
*Stock Solution (1.6 g/ml)
**Mix 25 g avertin and 15.5 ml tert-amyl alcohol at room temperture for ~12 hours in a dark bottle.
**Can store stock solutions at room temperature for one year.
*Working solution (20 mg/ml)
**Mix 0.5 ml avertin stock solution and 39.5 ml 0.9% saline in dark/foil covered container.
**Filter solution through 0.2 micron filter into a dark/foiled covered container.
**Store solution at 4 degree C.
**Replace working solution each month.
*Notes:
**Avertin is light sensitive.
**Degredation products are lethal.
**Always store at 4 degree C
**Do NOT use a solution that is yellow or contains a precipitate.
**Avertin is lipid soluble and may require a larger dose.

==Perfusion Pump Set Up==
*Place 30 ½ gauge on end of tubing
*Insert one tube into 9% saline solution, and one tube into 10% formalin solution
''Image 1: Set up of the two tube system from 0.9% saline solution and 10% formalin solution.''
[[File:perfusionbottle.png]]
*Turn pump on to a flow rate of 0.72 ml/min
*Have stopcock turned to allow for flow of saline
''Image 2: Stopcock set up to allow for 0.9% saline and 10% formalin solution to flow through to main line.''
[[File:perfusionstopcock.png]]
*Fill line with 0.9% saline until you have a steady stream of 0.9% saline from the end of the needle.
*Turn off peristaltic pump and turn stopcock to allow for formalin solution to flow
*Turn on peristaltic pump and pump 10% formalin solution until flow fluid is before the stopcock
**Flow has not entered the main line
*Turn off peristaltic pump and turn stopcock to allow for saline to flow
*Turn on pump and pump saline through the main line at stated flow rate
''Image 3: Overview of the entire perfusion setup (excluding the procedural platform in the collecting dish)''
[[File:perfusionsetup.png]]

==Animal Procedure==
*Place mouse in isofluorane chamber and wait for slowing of heart beat
**Approximately 1 heartbeat/second
*Remove mouse from chamber
*For mouse anesthesia, administer 0.8 ml/20 g (of mouse body weight) Avertin through intraperitoneal injection with 30 ½ gauge needle.
*Wait for 3 minutes or until the mouse no longer responds to painful stimuli, such as paw pinch before proceeding.
*Lay the mouse on its back.
*Using tweezers and operating/dissecting scissors open up the skin and expose the chest cavity.
*Cut open the diaphragm using standard scissors.
**Be careful to not pierce the heart.
*Using clamp scissors, grab at the base of the sterum and lift to exposure the heart.
*Transfer the mouse to the procedural stage/platform.
*Insert the 30 ½ gauge needle (from the tubing with saline/10% formalin solution) into the apex of the left ventricle.
*Immediately after inserting the needle into the left ventricle, cut the aorta using standard scissors.
**This allows for the blood to flow out of the mouse and drain into collecting dish.
*Perfuse with saline solution for 10 minutes.
*After 10 minutes, switch the stopcock to allow for flow of formalin solution, at same flow rate as saline.
*Perfuse for 10-12 minutes with 10% formalin solution.
*With the pump still on, remove needle from left ventricle.
**Mouse is now fixed for tissue collection.
*When collecting tissues, keep tissues in 10% formalin solution for ~24 hours after collecting and place in 4 degree C.
*Transfer tissues to PBS after for long-term storage at 4 degree C.

Perfusion of Mouse

2015-03-27T17:34:48Z

Aragaus1:

==Materials==
*0.9 % Saline solution
*10% formalin solution
*Avertin
*Isofluorane
*30 ½ gauge needle
*1 mL syringe
*Peristaltic pump
*Collecting dish
*Surgical stage/platform
**Make sure this is level with the edge of the collecting dish to allow for the need to rest during the procedure
===Surgical tools:===
*Dissecting scissors
*Forceps
*Clamp scissors
*Standard scissors

===Avertin Preparation===
*Taken from: http://web.jhu.edu/animalcare/rdf/avertin.html
*Stock Solution (1.6 g/ml)
**Mix 25 g avertin and 15.5 ml tert-amyl alcohol at room temperture for ~12 hours in a dark bottle.
**Can store stock solutions at room temperature for one year.
*Working solution (20 mg/ml)
**Mix 0.5 ml avertin stock solution and 39.5 ml 0.9% saline in dark/foil covered container.
**Filter solution through 0.2 micron filter into a dark/foiled covered container.
**Store solution at 4 degree C.
**Replace working solution each month.
*Notes:
**Avertin is light sensitive.
**Degredation products are lethal.
**Always store at 4 degree C
**Do NOT use a solution that is yellow or contains a precipitate.
**Avertin is lipid soluble and may require a larger dose.

==Perfusion Pump Set Up==
*Place 30 ½ gauge on end of tubing
*Insert one tube into 9% saline solution, and one tube into 10% formalin solution
''Image 1: Set up of the two tube system from 0.9% saline solution and 10% formalin solution.''
[[File:perfusionbottle.png]]
*Turn pump on to a flow rate of 0.72 ml/min
*Have stopcock turned to allow for flow of saline
''Image 2: Stopcock set up to allow for 0.9% saline and 10% formalin solution to flow through to main line.''
[[File:perfusionstopcock.png]]
*Fill line with 0.9% saline until you have a steady stream of 0.9% saline from the end of the needle.
*Turn off peristaltic pump and turn stopcock to allow for formalin solution to flow
*Turn on peristaltic pump and pump 10% formalin solution until flow fluid is before the stopcock
**Flow has not entered the main line
*Turn off peristaltic pump and turn stopcock to allow for saline to flow
*Turn on pump and pump saline through the main line at stated flow rate
''Image 3: Overview of the entire perfusion setup (excluding the procedural platform in the collecting dish)''

==Animal Procedure==
*Place mouse in isofluorane chamber and wait for slowing of heart beat
**Approximately 1 heartbeat/second
*Remove mouse from chamber
*For mouse anesthesia, administer 0.8 ml/20 g (of mouse body weight) Avertin through intraperitoneal injection with 30 ½ gauge needle.
*Wait for 3 minutes or until the mouse no longer responds to painful stimuli, such as paw pinch before proceeding.
*Lay the mouse on its back.
*Using tweezers and operating/dissecting scissors open up the skin and expose the chest cavity.
*Cut open the diaphragm using standard scissors.
**Be careful to not pierce the heart.
*Using clamp scissors, grab at the base of the sterum and lift to exposure the heart.
*Transfer the mouse to the procedural stage/platform.
*Insert the 30 ½ gauge needle (from the tubing with saline/10% formalin solution) into the apex of the left ventricle.
*Immediately after inserting the needle into the left ventricle, cut the aorta using standard scissors.
**This allows for the blood to flow out of the mouse and drain into collecting dish.
*Perfuse with saline solution for 10 minutes.
*After 10 minutes, switch the stopcock to allow for flow of formalin solution, at same flow rate as saline.
*Perfuse for 10-12 minutes with 10% formalin solution.
*With the pump still on, remove needle from left ventricle.
**Mouse is now fixed for tissue collection.
*When collecting tissues, keep tissues in 10% formalin solution for ~24 hours after collecting and place in 4 degree C.
*Transfer tissues to PBS after for long-term storage at 4 degree C.

File:Perfusionstopcock.png

2015-03-27T17:30:19Z

Aragaus1:

Perfusion of Mouse

2015-03-27T17:29:57Z

Aragaus1:

Perfusion of Mouse

2015-03-27T17:26:12Z

Aragaus1: Created page with "==Materials== *0.9 % Saline solution *10% formalin solution *Avertin *Isofluorane *30 ½ gauge needle *1 mL syringe *Peristaltic pump *Collecting dish *Surgical stage/platform..."

File:Perfusionbottle.png

2015-03-27T17:25:24Z

Aragaus1:

Main Page

2015-03-27T17:11:00Z

Aragaus1: /* Tissue Preparation */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]
*[[H&E Staining of Mouse Tissue]]
*[[Perfusion of Mouse]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

H&E Staining of Mouse Tissue

2015-03-27T16:16:58Z

Aragaus1:

==Materials==
*xylene
*100% Ethanol
*70% Ethanol
*H2O
*Mayer's Hematoxylin
*1% Eosin Y (in water)
*95% Ethanol

==Protocol==
*Removing Wax and Rehydrating
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
# Deparaffinize and rehydrate sections
## Immerse for 5 min with agitation in xylene
## Immerse for 5 min with agitation in 100% ethanol
## Immerse for 5 min with agitation in 70% ethanol
## Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min
*H&E staining
#Dip slide into Mayer's Hematoxylin and agitate for 30 sec
#Rinse slide in H2O for 1 minute
##depending on the intensity desired, repeat this step if necessary
#Dip slide in 1% eosin Y fo 10-30 sec with agitation
*Dehydration
#Place slides in 95% Ethanol two times for 30 seconds each.
#Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
#Wash twice with fresh xylene for 30 seconds.
#Let slide dry.
#Place 1-2 drops of mounting medium on slide and place coverslip over
#Seal coverslip with sealant(nailpolish)

==Notes==
*Be sure to shake Eosin prior to placing slide in for staining because it will settle.
*The staining will get more intense after time, be sure not to oversaturate section

*Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986

H&E Staining of Mouse Tissue

2015-03-27T14:48:52Z

Aragaus1: Created page with "==Materials== *xylene *100% Ethanol *70% Ethanol *H2O *Mayer's Hematoxylin *1% Eosin Y (in water) *95% Ethanol ==Protocol== *Removing Wax and Rehydrating # Place slide on ed..."

==Materials==
*xylene
*100% Ethanol
*70% Ethanol
*H2O
*Mayer's Hematoxylin
*1% Eosin Y (in water)
*95% Ethanol

==Protocol==
*Removing Wax and Rehydrating
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
# Deparaffinize and rehydrate sections
## Immerse for 5 min with agitation in xylene
## Immerse for 5 min with agitation in 100% ethanol
## Immerse for 5 min with agitation in 70% ethanol
## Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min
*H&E staining
#Dip slide into Mayer's Hematoxylin and agitate for 30 sec
#Rinse slide in H2O for 1 minute
##depending on the intensity desired, repeat this step if necessary
#Dip slide in 1% eosin Y fo 10-30 sec with agitation
#Wash twice with fresh xylene for 30 seconds.
#Let slide dry.
#Place 1-2 drops of mounting medium on slide and place coverslip over
#Seal coverslip with sealant(nailpolish)

==Notes==
*Be sure to shake Eosin prior to placing slide in for staining because it will settle.
*The staining will get more intense after time, be sure not to oversaturate section

*Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986

Main Page

2015-03-24T19:43:34Z

Aragaus1: /* Tissue Preparation */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]
*[[H&E Staining of Mouse Tissue]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Main Page

2015-03-24T19:43:23Z

Aragaus1: /* Tissue Preparation */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]
*[[H&E Staining of Mouse Tissue}}

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Paraffin Embedding of Tissue Samples

2015-02-18T17:51:11Z

Aragaus1: /* Paraffin embedding protocol */

== 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!).
#Turn on wax pots.
#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 Citrosolv for 30-60 minutes. Repeat this step a second time, using fresh Citrosolv.
#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.

Wash Solution Table
{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"
|-
!style="border-style: solid; border-width: 0 1px 1px 0"| Solution
!style="border-style: solid; border-width: 0 0 1px 0"| Time
|-
|style="border-style: solid; border-width: 0 1px 0 0"| 70% ethanol
|style="border-style: solid; border-width: 0"| Overnight/24 hours
|-
|style="border-style: solid; border-width: 0 1px 0 0"| 75% ethanol
|style="border-style: solid; border-width: 0"| 30 min
|-
|style="border-style: solid; border-width: 0 1px 0 0"| 95% ethanol
|style="border-style: solid; border-width: 0"| 2x 75 min
|-
|style="border-style: solid; border-width: 0 1px 0 0"| 100% ethanol
|style="border-style: solid; border-width: 0"| 3x 60 min
|-
|style="border-style: solid; border-width: 0 1px 0 0"| Citrosolv
|style="border-style: solid; border-width: 0"| 2x 30 min
|-
|style="border-style: solid; border-width: 0 1px 0 0"| 60 degree paraffin
|style="border-style: solid; border-width: 0"| 3x 60 min
|}