[Histonet] IHC pretreatment with NaOH/H2O2

Neil M. Fournier neil.fournier <@t> yale.edu
Thu Feb 17 23:53:30 CST 2011

I recently came across a protocol that was conducting IHC staining for pSTAT3 on
free-floating fixed rat brain sections. The protocol stated that the tissue was
placed in 1% NaOH and 1% H2O2 in H2O for 20 min, 0.3% glycine for 10 min, and
0.03% sodium dodecyl sulfate for 10 min.

Could someone please explain to me the rationale for using NaOH and H2O2
pretreatment, as well as the additional steps in this procedure? My rationale
may be incorrect but I would be concerned that the combination of NaOH and H2O2
might produce a too much O2 bubbling that could harm the tissue. I believe
glycine is typically used for reduce autofluoresence since it binds to free
aldehydes but this protocol was utilizing a cobolt/nickel enhanced-DAB reaction
and I am unaware of any empirical study that has shown that glycine pretreatment
improves peroxidase immunostaining. Finally, SDS seems like a fairly harsh
detergent (although the remaining protocol utilizes Triton X-100 in blocking
and antibody incubation steps), has anyone used this? and what would be the
benefit of this over other detergents.

Thanks in advance


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