[Histonet] Cryosectioning, fixing and H&E staining
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
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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