Neil, When I read the paper in question, it reminded me of the pretreatments one would use in an In Situ Hybridization protocol. I have used this protocol in the past and it doesn't seem to harm the tissue as long as the tissue has been adequately perfused. We use the pSTAT3 #9131 (Cell Signaling) with 40um free-floating rat brainstem sections that have been perfused with 4% paraformaldehyde .
After successfully reproducing their protocol, I decided to try and incorporate my own protocol for this particular antibody. The only pretreatment that I use is a cocktail of Methanol (cold) + 1% H2O2 for 20 minutes., TBST Rinses, Blocking step - Rodent Block R, Primary - pSTAT3 (1:50 - 1:100) - overnight, TBST Rinses, Alexa Fluor secondary - 2hrs., TBST Rinses, mount & coverslip with ProLong Gold (Invitrogen). **The #9131 spec sheet notes that you must use Methanol as a pretreatment step for this anitbody. *** I am often required to use this antibody in double labeling experiments with tract tracing beads that are sensitive to heat. HIER is out of the question, but the methanol/H2O2 pretreatment appears to be adequate for this antibody and doesn't affect the beads. Regards, Tina Montina J. Van Meter Lab Manager Pennington Biomedical Research Center Autonomic Neuroscience -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Johnson, Teri Sent: Friday, February 18, 2011 1:07 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Re: IHC pretreatment with NaOH/H2O2 Dear Neil, I was able to find a protocol like you described published where they had done a regular IHC protocol on free-floating tissue sections for a-MSH antibody using 0.3% H2O2, normal serum block, etc. They go on to describe that for P-STAT3, "the tissue needed to be pretreated with 1% NaOH and 1%H2O2 in water for 20 minutes, 0.3% glycine for 10 minutes, and 0.03% SDS for 10 minutes". After that it looks like a standard protocol. Here's the URL for that paper: http://endo.endojournals.org/cgi/reprint/144/5/2121?ijkey=23f435f1cf6162 9953465217ca752dd73df366e9 They don't say why this was necessary, but it might be worth trying to track down one of the authors and ask them. It seems like the sodium hydroxide and hydrogen peroxide serves as an oxidizer somehow. Since they are not doing IF, maybe the glycine is needed to bind with glycine receptors in the brain prior to staining? And I suspect the SDS is needed for permeabilization, even though they are using a sectioned sample. The right detergent can make all the difference. I would really be interested in knowing what the rationale is, but it seems like they figured out what physiologic changes needed to happen for this antibody to work in brain. If you do have a conversation with the authors, please report back here because my curiosity is now at high roar. Best wishes, Teri Johnson, HT(ASCP)QIHC Head, Histology and Electron Microscopy Stowers Institute for Medical Research Kansas City, MO _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet