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| | | == Paraffin embedding protocol == |
| == Protocol 1 ==
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| #After harvesting tissue from animal, place into pathology cassette and fix in 4% formaldehyde, 4% paraformaldehyde or 4% formalin O/N
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| #After fixation, begin dehydrating the tissue in 70% Ethanol
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| #On the day of planned embedding, wash sample with 100% Ethanol 3 times for 30 minutes.
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| ##While washing in 100% Ethanol, start the paraffin wax machine (Link Building, Room 311)
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| #After 100% Ethanol washes, Wash 3 times in Xylenes
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| #Wash samples in a beaker filled with wax at 58-60C, 3 times for 15 mins
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| #Immediately before embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with wax
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| #Take your sample and orient it on top of the base wax as desired
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| #Place pathology cassette on top of the mold and fill with wax covering the mold
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| #Place mold with sample on "Cold Side" of embedding station
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| ##Wax will solidify in 10-15 minutes
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| #Remove wax embedded sample from the mold (should just slip out)
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| #Store at room temperature until prepped to section
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| == 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). | | #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. | | #Move cassettes into 70% ethanol for 24 hrs. Samples can be left in 70% for long term storage, if required. |
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| #Remove embedded sample from the mold (should just slip out). | | #Remove embedded sample from the mold (should just slip out). |
| #Store at room temperature until ready to section. | | #Store at room temperature until ready to section. |
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| Protocol Edited from: FFPE SOP - MKM and SR for the Williams Lab
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| 1. Collect tissue
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| a. Immediately place in labeled cassette in 10% buffered formalin
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| b. Place in labeled cassette on dry ice prior to storage at -80C
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| 2. Fix in 10% buffered formalin
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| a. NOTE: for 3 mm mouse brain sections NEVER exceed 24H in formalin
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| 3. Tissue processing
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| a. 70% EtOH (30 min) → tissues can be kept in 70% Ethanol for long-term storage
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| b. 95% EtOH (30 min)
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| c. 100% EtOH (30-60 min)
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| d. Xylene (30 min under a fume hood)
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| e. Xylene (30 min under a fume hood)
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| f. Rinse tissues in Paraffin (melting temp of wax, 56 oC for paraplast X-tra)
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| g. Paraffin (melting temp of wax, 56 oC for paraplast X-tra)
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| *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.
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| 4. Embedding
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| a. Spray the stainless steel cassette with ‘base mold release agent’ prior to adding heated wax.
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| 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.
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| c. Place the back side of the plastic labeled cassette on top of the stainless steel cassette and fill until wax just covers mesh.
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| d. Chill until paraffin is completely solidified and then remove stainless steel cassette.
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| Sectioning
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| 1) Section 10-15 micron sections from cold blocks of paraffin imbedded tissue with cold blade (pre-chilled in 4 oC).
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| 2) Pretreatment of paraffin tissue sections with either of the following
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| a. Leave slides at room temperature for 60 minutes; or
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| b. Heat in dry oven at 55°C for 20 minutes
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| Note 1: for adipose, leaving at room temperature prevents disruption of the tissue
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| Note 2: For RNA and antigens option (b) is recommended. Option (a) is for highly heat sensitive molecules.
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| 3) Immerse slide in xylene for 2 min. (fat) or 10 min. (liver).
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| 4) Immerse slide in 100% ethanol for 2 minutes.
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| 5) Immerse slide in 95% ethanol for 1 minute.
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| 6) Immerse slide in 70% ethanol for 1 minute.
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| 7) Immerse slide in 50% ethanol for 1 minute.
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| 8) Immerse slide in 1X PBS for 2 minutes.
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| 9) Air dry slides for 10-20 min
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| Protocol Edited from: several online IHC protocols
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| H&E Staining
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| Hematoxylin staining
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| Incubate slides in Mayer’s Hematoxylin for 20-30 min (fat) or 10 min (liver)
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| Rinse in warm H2O (note ‘blueing’ of nuclei)
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| Eosin staining
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| Working solution:
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| • 25 mL of 1% Eosin (dissolved in 2 parts of ddH2O and 8 parts of 95% Ethanol)
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| • 75 mL of 80% Ethanol
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| • 0.5 mL Glacial Acetic Acid
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| Stain sections for 10 min. in 0.25% Eosin solution
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| Rinse slides in ddH2O
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| Dehydrate slides in
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| 1) Immerse slide in 70% ethanol (4 dips)
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| 2) Immerse slide in 95% ethanol (2 dips).
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| 3) Immerse slide in 100% ethanol (2 dips).
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| 4) Immerse slide in xylene for (6 dips)
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| Mounting
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| Apply a drop of mounting medium over the section
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| Place the cover slip over in an angle to avoid bubbles
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| Store slides at room temperature for 2 h prior to analysis
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