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Haematoxylin Eosin staining for frozen sections - H&E staining frozen sections (Nov/02/2004 )


Does anyone of you has a protocol for H&E staining for frozen sections? Is this different than staining paraffin sections?



normally one can't make anything wrong with HE staining. Take mayers Hämatoxilin-solution, put as much as you want on the slide, wait as long as you want and hold it under water as long as you want. its recommended to take tap water, because its normally a little bit basic (pH8). eosin I don't take, for lymphocyte infiltrates etc häm. is enough.


Are the sections fixed frozen or fresh frozen?


-- Take section immediately from freezer (do not let dry on benchtop) and submerge in fixative -- 10% Neutral Buffered Formalin for 10 minutes
-- Wash well in several changes of PBS to remove OCT or other tissue embedding compound then rinse in water
-- Incubate in Harris' hematoxylin for 5 minutes (perhaps less or more depending on your tissue and batch - check it out), followed by copious rinses in dH20
-- Differentiate in acid alcohol (1% HCl in 70% EtOH), 1-2 dips - check under scope to make sure is ok Rinse well in dH20
-- Blue in Ammonia water (0.25% ammonia hydroxide in dH20) or other basic bluing solution (including lukewarm tap water), followed by rinses in dH20
-- Dehydrate to 95% ethanol
-- Dip for 10-20 seconds in alcoholic Eosin
-- Proceed through at least 2 more changes of 95% ethanol, 100% ethanol, xylenes.
-- Coverslip.


Here is my question...
After cutting the frozen section, should I fix that immediately and proceed later with H&E staining???
I put them at 4 degrees after cutting to H&E later. Is that ok?



there really is no difference between frozen and parafin sections once the oct or wax has been removed however as wax sections tend to be fixed in formalin they may need epitope retrieval to counteract the cross linking. as frozen sections are unlikely to survive boiling in the microwave (they wont have been baked on) i would recomend you fix in 50:50 methanol : acetone.

after cutting frozen sections wrap them in saran and stick them in a -80 freezer or if you are staining the same day they'll be ok unfixed for an hour or so but the sooner you fix the less chance of degredation.
the 4 degrees wont really make much of a difference - defrosted is defrosted.