Re: NF-KB problems
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From: | Masayuki Miyagishima <mmiyagis+@pitt.edu> (by way of histonet) |
To: | histonet <histonet@magicnet.net> |
Reply-To: | |
Content-Type: | text/plain; charset="us-ascii" |
Here is the protocol of NF-kB staining, which ended up with just
interstitial staining( no stain inside cell).
We fixed Tissue with 2X parafolmaldehyde for 2 hours and 30 % sucrose
overnight before freezing in isopentane. Then 10#027#(I5#027#(Jm frozen
sections were
treated with 5 % normal goat serum for 1 hour at RT, washed with PBS/BSA
and incubated with sc 372 anti-p65 antibody for 1 hour at RT, then
washed and incubated with goat anti rabbit fit C antibody for 1 hour at
RT.
Should I do antigen retreival, and prolong the time of incubation?
--
Masayuki Miyagishima, MD
University of Pittsburgh,
Department of Surgery
412-647-2345, and ask the hospital operator to page 3228, please.
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