[Histonet] Re: zincTris fixation on frozen sections

Gayle Callis gcallis <@t> montana.edu
Wed Apr 6 10:07:32 CDT 2005


We have tried what you want to do, without success.

For answer to question #1 - IF you try this, NO you would just rinse in 
TRIS buffered saline X 3 after ZnTRIS fixation. You should do a  time panel 
to determine if this is going to work, 2,4,6,8,10, 15, 30 minutes - 
whatever.  I have never seen this fixative used for fresh frozen sections, 
only tissues destined for paraffin embedding.  Further comments next.

For answer to question #2, when we tried ZnTRIS on fresh frozen sections 
murine tissue for CD markers, it was a total failure.   Morphology was no 
better and IHC staining was poor and diffuse. We only do solvent fixation 
for surface markers.  If we used ZnTRIS , it would only be for paraffin 
work to avoid NBF for CD markers. Although touted to have good morphology 
with paraffin sections, we have not found that to be true, plus the mouse 
tissues were dry, friable and difficult to cut.   We did get staining of CD 
markers though.  We found it much faster with better results to do use the 
following suggestions.

You can try other fixation protocols to try and improve the morphology

1.  An acetone/alcohol fixation (AA)  Air dry FS overnight at RT, they MUST 
BE VERY DRY.  Fix next day (of staining) in 75% acetone/25% absolute 
ethanol or 75 ml acetone mixed with 25 ml 100% ethanol (do NOT use methanol 
or any other alcohol) .  Fix for 5 minutes, then go directly to buffer 
rinse.  DO NOT air dry sections again, and proceed to staining.

2.  A double acetone fixation protocol works also, although morphology is 
not as good as AA fixation.  We rarely do single acetone fixation, and if 
we do it, very dry FS in 4C acetone for 10 min, air dry and stain.  The 
double acetone protocol is: Air dry FS overnight at RT (over 16 mesh silica 
gel), then fix in 4C acetone for 10 min, air dry 15 min in front of fan if 
possible, go back to acetone for 10 min at 4C, air dry 20 min and proceed 
with staining.

It is a dilemma, but we live with some loss of morphology and excellent IHC 
staining for all our  cell surface markers, an unfortunate circumstance 
when working with certain surface markers in the mouse.  Overall, our 
sections with AA are excellent, although some surface markers may not like 
the alcohol component, and others may like only minimal fixation in cold 
acetone - a lot depends on the antigen.  We have even found CD markers that 
like AA better than vendor suggested cold acetone fixation, although they 
say it will not work.  Rule is TRY IT!!!

At 09:22 PM 4/5/2005, you wrote:
>I am in the process of staining mouse kidney samples containing grafted 
>mouse pancreatic islets with antibodies against MHC H2Dd and H2Db. Tissue 
>samples available are either FFPE or unfixed, snap frozen. I decided to 
>concentrate on the frozen samples due to the general difficulty of 
>staining for surface markers after formalin fixation. My questions are:
>1. If I want to use frozen sections, immediately fixed in zincTris 
>(formalin-free), will I need to process the slides through increasing 
>alcohols just as suggested for zincTris fixation of tissue samples ?
>2. Will zincTris fixation yield better morphology compared to conventional 
>acetone fixation ?
>Yves Heremans
>University of Minnesota
>Stem Cell Institute
>Tel 612-625-0964
>Fax 612-624-2436

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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