NF-kB staining in frozen mice heart

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From:"Masayuki Miyagishima, MD" <>
To:HistoNet <>
Date:Fri, 12 Mar 1999 23:33:13 -0500
Content-Type:text/plain; charset=iso-2022-jp

We have been trying to stain NF-kB (p65) in mice heart.  We got sc372
rabbit anti mice, rat p65 antibody from Santa Cruz, and using secondary
Goat anti-rabbit FitC antibody.  We refix frozen tissue with 2%
paraphormaldehyde and treat with normal goat serum overnight at 4 C and
primary overnight at 4 C, then overnight secondary at 4 C.  We dont see
positive staining (Nuclear staining) in these tissue.  We tried with
cell treated with IL1b and still get few staining.   We tried changing
fixation to Aceton, Methanol, etc, or Saponin treatment, etc, but failed
to improve the result.  Do you think I should consider getting another
antibody at this stage?#027#$B!!!!#027#(JAny recommendation for mice frozen tissue
immunohistochemistry?  I would appreciate any imput....
Masayuki Miyagishima, MD
University of Pittsburgh, 
Department of Surgery

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