Paraffin Embedding of Tissue Samples: Difference between revisions

Newer edit →

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 #Remove wax embedded sample from the mold (should just slip out)
 #Store at room temperature until prepped to section
+Protocol Edited from: FFPE SOP - MKM and SR for the Williams Lab
+.	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
+.	Fix in 10% buffered formalin
+a.	NOTE: for 3 mm mouse brain sections NEVER exceed 24H in formalin
+.	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.
+.	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
+)	Section 10-15 micron sections from cold blocks of paraffin imbedded tissue with cold blade (pre-chilled in 4 oC).
+)	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.
+)	Immerse slide in xylene for 2 min. (fat) or 10 min. (liver).
+)	Immerse slide in 100% ethanol for 2 minutes.
+)	Immerse slide in 95% ethanol for 1 minute.
+)	Immerse slide in 70% ethanol for 1 minute.
+)	Immerse slide in 50% ethanol for 1 minute.
+)	Immerse slide in 1X PBS for 2 minutes.
+)	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
+)	Immerse slide in 70% ethanol (4 dips)
+)	Immerse slide in 95% ethanol (2 dips).
+)	Immerse slide in 100% ethanol (2 dips).
+)	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