[Histonet] Problems with immunohistochemistry on formalin-fixed heart sectio ns

[Barlow, Gillian]

Dear Histonetters

This is the latest installment in our ongoing heart saga, I hope someone out
there can help!!!

Following advice from you all (many thanks!) we now follow the following
protocol:  we harvest our hearts from live but anaesthetized animals so that
they are still beating, cut them in half to allow the fixative access to the
tissues, and fix in 10% buffered formalin for usually 12 to 24 hours at room
temp.  After this, we process, embed in paraffin and section at 8 microns.
The preservation of the tissue looks good when we stain with H&E, but when
we do the immunohistochemistry, the cells look destroyed.  Commercial
sections purchased from Novagen, who fix their hearts in 4%
paraformaldehyde, look fine using identical immunohistochemical techniques.
Are formalin-fixed hearts known to be more sensitive e.g. to pretreatment
with peroxide to block endogenous peroxidases?  Or is there some other step
likely to be causing trouble? The antibodies are from Santa Cruz, and are
supposed to work well on formalin- or PFA-fixed tissues.

All suggestions gratefully received!

Many thanks
Gillian

Gillian Barlow, PhD
Postdoctoral Fellow
Laboratory of Julie Korenberg, PhD, MD
Cedars-Sinai Medical Center
Davis Bldg, Lab 2007
110 George Burns Rd
Los Angeles, CA 90048

Phone: (310) 423 7650
Fax: (310) 423 0302
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