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

From:CM Bush (by way of histonet)

Hi Lorie,

Thanks for getting back to me.
Yes this tissue has been fixing in 10% NBF and cut on a freezing stage
microtome (not a cryostat).  This tissue has been in 10% NBF for
month-years, so the tissue is very well fixed.  Right, I guess I should
have emphasized this in my post.
As well I left out, the reason we are cutting the brain tissue on the
freezing stage microtome, is so that we may cut thick (40um) sections,
which will flatten out on the slides (thick paraffin sections wrinkle more
than will work for what we are doing.)

Clarissa wrote:
Why are you doing antigen retrieval on frozen sections? AR is for paraffin
tissues that are fixed ... or are your frozens fixed as well?


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-----Original Message-----
From: CM Bush 
Sent: Fri, 14 Oct 2005 15:00:08 -0700 (PDT)
Subject: [Histonet] IHC in Fixed Frozen vs. Processed and Paraffin Embedded
Brain Tissue

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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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