[Histonet] question about H2O2 use during immunohistochemical protocols

From:Neil Fournier

Hi everyone,

We work with 40 to 50 micron thick fixed (w/ 4% paraformaldehyde) rat brain sections that had been sectioned on a vibrating microtome. The sections are stored for varying lengths of time in a cryoprotectant solution (consisting of sucrose/PVP-40/ethylene glycol in 0.1 M PBS. Watson et al., 1986. Peptides) at -20 degree C. 

After a thorough washing of the tissue sections several times in PBS rinses, the sections are incubated in 0.3% H2O2/PBS solution (diluted from 30% stock) for 30 to 60 min (depending on protocol). We use Netwells to transfer tissue sections from wash to wash etc. I have noticed that often excessive bubbles form in the culture wells that contain the tissue sections when incubated in the peroxide mixture.  At first we thought our stock of previously aliquot 30% H2O2 might have went bad, so I purchased new chemicals and we still observe this bubbling problem.  We do not know how this might impact staining quality or if these bubbles might even cause some degree of microscopic damage to the section, but we have noticed lately that some sections show incomplete penetration of the antibody during staining (i.e., staining appears darker for one part of the tissue and lighter for other parts). I do not remember encountering this before. I do not believe it is related to the cryoprotectant b/c this solution is commonly used in several laboratories and I often do around an 1 hr of 5 to 10 min washes before beginning the protocol. 

I was wondering if anyone here has encountered similar situations when staining tissue and if they know whether this is a problem to staining and what the exact cause might be.

Thanking everyone in advance,

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