Re: rat/mouse H&E staining

From:Gayle Callis

Addtional comments about rat/mouse H&E staining.

The H&E staining information I gave Ron was for 5 um thick formalin fixed
paraffin sections (FFPE), and when staining fresh frozen sections, I cut FS
section, 5 um, and go immediately into neutral buffered formalin for no
less than 30 seconds, no air drying.  Frozens can be stored in NBF until
one is ready to stain - up to weeks. NBF improves fresh frozen section
morphology tremendously for H&E. In a pinch and a hurry, 95% ethanol works,
but sections tend to appear a bit more dessicated/ragged.  

In general, we have found fresh frozen NBF fixed H&E staining very close to
timing of our paraffin section staining and with similar good results. 

Question, in this message:  are your brain/spinal cord tissues PREFIXED
with paraformaldehyde or anything before you do the frozens?  since you
indicated going down through xylene, alcohols, etc to do staining. I'm
confused here??  If they are fresh frozens, and you do this, then alcohols
in rehydration protocol must be doing fixation, not sure what xylene does
with fresh frozens, any comments??  I can understand the problem with
thicker sections.  


 09:58 AM 7/24/02 +1000, you wrote:
>We had some trouble staining our rat brain and spinal cord sections
>(30Ám)  with H&E (Usually we use cresyl violet). I am assuming you
>are talking about frozen tissue sections. Our biggest problem was
>patchy staining, and that the thicker sections were too dark. In the
>end we used the standard H&E run used for parafin sections, Harris
>Haematoxylin (regressive) and eosin but with reduced times (esp for
>eosin) and careful differentiation in acid alcohol.
>I can't help with exact times as they differ so much between
>batches/types of H&E.
>The other thing we were finding was that we got much more even
>results if the air dried sections were taken down through xylene and
>alcohol in exactly the same way you would for parafin sections. If
>you are using parafin embedded sections there should be no difference
>in the staining for rat/mouse tissue to what you might usually use in
>the clinical settings.
>Cheers, Cath
>At 9:16 AM -0400 22/7/02, Martin, Ronald wrote:
>>Fellow histotechs,
>>If anyone is willing to share their H&E staining procedure for rat/mice
>>tissue I would greatly appreciate it. I am staining brain, skin, sub
>>cutaneous  tumors, stomach and whole 1 day olds. I would like to know what
>>type of stains (brands), times and techniques (progressive or regressive)
>>some of you are using. Once again, this is for rat/mice tissue in a research
>>setting, not a clinical setting.
>>Ron Martin, B.Sc., HT/HTL (ASCP)
>Cathy Gorrie
>Scientific Officer
>Neural Injury Research Unit,
>Department of Anatomy
>School of Medical Sciences,
>University of New South Wales
>Sydney, N.S.W. 2052
>Phone: 61-2-9385 2462
>Fax   : 61-2-9313 6252
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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