Re: [Histonet] IHC in Fixed Frozen vs. Processed and Paraffin Embedded Brain Tissue

From:Rene J Buesa (by way of histonet)

In our laboratory all the frozen sections destined for IHC were air dried,
acetone fixed.
The OCT was dissolved in water afterwards, the slides placed in PBS and we
did NOT
use HIER on them.
The objective of HIER, as you perfectly know, is to "undo" the crosslinking
consequence of formlin fixation, but if you don't fix in formalin, you
don't need HIER.
Why don't you try this approach? For us always worked OK.
Rene J.

Patti Loykasek  wrote:
If you are performing the HIER in the microwave pressure cooker on the
frozen sections, I think perhaps you are "over-retrieving" those sections.
That is a harsh pretreatment to apply to frozen sections. We've had luck on
frozen sections that we cut on a cryostat, fix in 10%NBF for 30-60 minutes,
retrieve in citrate in a 75 degree water bath for 30 minutes, then perform
the IHC. I can't be positive that this will fix your problems, but just
something we've tried that works for us. I will say we have had some trouble
with calretinin on FS tissue, too.

Patti Loykasek BS, HTL, QIHC
PhenoPath Laboratories
Seattle, WA

> Hello there Dear Histonet,
> I am re posting this from a few days ago. The were no responses, so I'm
> hoping maybe this time. Much thanks for everyone's patience.
> We are trying to stain human anterior cingulate brain with anti
> Calretinin. We have taken a sample of said tissue (fixed in 10% NBF);
> processed and paraffin embedded one portion of the tissue and cut the other
> portion on a freezing stage microtome (fixed frozen sample cryo protected
> with 30% sucrose). (The sections for both varieties mounted on slides.)
> We have been able to see Calretinin staining (at 1:3000) for the
> processed, embedded tissue but not for the fixed frozen version of the same
> tissue. For all tissue, antigen retrieval was done using citrate
> buffer pH6 and microwaving 12 min high and 6 min low, in a pressure cooker.
> Also, we used TBS buffer with 0.4% Triton X for all sections.
> Would anyone have any ideas as to what might be going on that the
> processed/embedded tissue will stain and the fixed frozen version of the
> same sample does not?
> Thank you very much, again.
> Clarissa
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