[Histonet] Collagen immunohistochemistry. Enzymatic or heattreatment of frozen sections.

Mon May 1 14:49:45 CDT 2006

Thanks, but what I would like to know is if you have to use the enzyme in unfixed sections as recommended by manufacturer or in samples already fixed in e.g. acetone. Usually we cut frozen tissue and fix sections in acetone. We then store them at -20 ºC util needed. 

  In fixed samples, is it possible to expose the antigenic site in collagen molecule that is recognized by the Ab?

  If you have to treat samples with hyaluronidase before fixing the tissue, then I would not be able to use the stored sections, and we would have to change our procedures so to enzymatically treat samples right after cutting tissue blocks, before fixing and storing them.  

  Best regards,

  Guillermo 

Patsy Ruegg <pruegg <@t> ihctech.net> escribió:
  The hylaronidase is to unmask the target site for the collagen antibody, the
hyalaronic acid region of the collagen epitope needs to be digested away so
you can get at the collagen 1 expression. It is not a fixative, it is more
like enzyme induced epitope retrieval (eier). I did find it necessary to
get good staining for some collagens.
Patsy 

Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 215
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
wk email pruegg <@t> ihctech.net
web site www.ihctech.net

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Guillermo
Palao
Sent: Monday, May 01, 2006 9:45 AM
To: Histonet
Subject: [Histonet] Collagen immunohistochemistry. Enzymatic or
heattreatment of frozen sections.

Hello all,

I am working with frozen sections to quantify collagen staining. I am
using Anti-Collagen-I Ab from Calbiochem, and they recommend fixing sections
after heating or enzymatic treatment with hylaronidase. I have a stock of
already acetone-fixed frozen sections where I have tried staining with
Anticollagen without success. Before staining I heated fixed sections for 10
min at 42 ºC. I now plan to try hyaluronidase treatment, but I would like to
know if anyone has experience staining sections with anti-Collagen once they
are already fixed with acetone without previous enzymatic or heat treatment.
What is the best way to do it? Is it necessary to cut new tissue and treat
it with heat or hyaluronidase before fixation?

As always thank to all you in advance,

Guillermo

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