[Histonet] Re: IHC pretreatment with NaOH/H2O2

Montina Van Meter Montina.VanMeter <@t> pbrc.edu
Fri Feb 18 13:50:26 CST 2011


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.



Montina J. Van Meter
Lab Manager
Pennington Biomedical Research Center
Autonomic Neuroscience

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Johnson,
Sent: Friday, February 18, 2011 1:07 PM
To: histonet <@t> 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:

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

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

Best wishes,

Teri Johnson, HT(ASCP)QIHC
Head, Histology and Electron Microscopy
Stowers Institute for Medical Research
Kansas City, MO

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