H&E stain on Re: [Histonet] cryosections

[Gayle Callis]

We do H&E on frozen sections frequently.   Cut frozen sections, immerse 
them immediately into NBF, you can fix for 15 minutes or longer (have 
waited days or even even weeks before H&E) then stain with a basically 
routine paraffin section H&E protocol. We have not fixed with Bouins 
though.   We do not air dry frozen sections destined for an H&E stain, they 
go directly to NBF.

Be careful to do tap water rinsing gently - FS are more fragile, use Plus 
Charge slides.

After fixation, rinse briefly to get rid of NBF and proceed with 
staining.   Stain is a progressive hematoxylin, Gill II or Richard Allen 
Hematoxylin 1 for 1 1/2 minutes, rinse 1 minutes, dip 20 dips in Richard 
Allen Clarifier, a mild acetic acid solution,  rinse in running tap water 1 
min, bluing is either Scotts tap water substitute (30 sec) or Richard Allan 
bluing solution for 30 sec to 1 min, rinse with running tap water 1 min, 
70% ethanol, eosin y for 30 seconds, dehydrate, clear and mount coverslip.

You can probably use any hematoxylin you like as long as you adjust timing 
for your frozen sections and we avoid ammonia water for bluing.  If your 
tap water is alkaline, you can rinse in warm tap water for 1 minutes for 
bluing also.



Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)