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

From:Patti Loykasek (by way of histonet)

    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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