[Histonet] Re: IHC pretreatment with NaOH/H2O2

[Johnson, Teri]

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=23f435f1cf61629953465217ca752dd73df366e9

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