[Histonet] Cryosectioning, fixing and H&E staining

From:Gayle Callis

We have good succes with fresh tissue frozen sections, cut and immerse 
immediately into neutral buffered formalin - fix for as long as you want, 
20 mintues, or even 10 min.  We often leave the FS in for a week on 
longer!  Mount your sections onto Plus Charge slides.  Make your running 
tap water rinses gentle!!  Rough handling of FS will knock them off the 
slide.  If you use strong Ammonium hydroxide to blue, the alkaline solution 
can eat sections off slides.  Use Scotts tap water substitute or Richard 
Allan bluing solution.  Destaining after hematoxylin is not going to adjust 
your eosin staining unless you do not rinse properly after bluing step.

Rinse section with tap water, distilled water rinse, Hematoxylin 1 (Richard 
Allan) for 1 1/2 min - progressive hematoxylin, not Harris variety.
Running tap water rinse 1 min
Clarifier (Richard Allan)  - 10 dips (fast!)
Rinse tap water 1 min
Bluing solution (Richard Allan) 1 min
Rinse tap water for 1 min
70% ethanol 1 min
Eosin Y 30 sec to 1 min (Richard Allan product) - normally frozen sections 
take up eosin readily.
95% X 2, 100X 2, clear and mount.  Rinses are 30 dips or more each.

Our frozen section H&E staining looks no different than our paraffin 
sections.  Your problem may be the pH of your eosin or adjusting the pH 
before you go into eosin.  The rinse after bluing is critical to remove any 
remaining cations, and the 70% alcohol rinse helps do that plus 
equilibrates the section with the same percentage of alcohol contained in 
eosin y solution.  In the past, we observed Gluteraldehyde fixed tissue 
stains with eosin differently as compared to NBF fixed tissue.

At 10:38 AM 7/30/2004, you wrote:
>      I want to cryosection collagen (bovine type I) matrixes seeded with
>human stromal fibroblasts.  I am looking at collagen production by the
>fibroblast.  My two questions are 1) Is it better to fix before or after
>cryosectioning?  I'm using a 1% glutaraldehyde- 10% formalin fixative.  2)
>After section I want to use a Hematoxylin (Modified Harris) and Eosin Y
>alcoholic stain, but I'm having trouble seeing the Eosin stain.  I have
>tried shortening the destaining step, but it doesn't seem to be working.
>Also, the staining steps, some of my samples don't want to stay on the
>Department of Mechanical Engineering
>University of Minnesota
>Histonet mailing list

Gayle Callis
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)

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