[Histonet] RE: Methanol in H2O2 explanation

[Teri Johnson]

Patrick Lewis wrote:

So what I'm gathering from all this.

Thanks by the way for the discussion.

Methanol is unnecessary for the H2O2, reaction, but it can make it slightly faster since both methods will be working in concert.   If you have tissue that has more peroxidase than usual, then extra time is required.

Also, If you use straight aq H2O2 on frozen sections the bubbling may damage the tissue.



I too am loving the discussion. It truly highlights what we continue to do due to tradition without thinking and how to back up and research where things started and why. It also highlights what we thought we knew and what we know now. Good stuff!

As for H2O2 on frozen sections, regardless of the solvent (MeOH vs buffer vs aq) we always brought the H2O2 concentration down by 10% (0.3% final) and increased the time to 30 minutes to avoid the tissue damage issue. Because of its known effect on some antigens, I discontinued using methanol many years ago for both paraffin and frozen section.

To my understanding, the idea of diluting 30% to working concentration allowed for more consistency vs buying 3% off the pharmacy shelf. Who knew how long the bottle had been sitting on the shelf? After practicing both options, I found no difference in efficacy in quenching endogenous peroxidase.

Some add sodium azide to the quenching solution because it is known to inhibit peroxidase and is believed to be a more "complete" peroxidase blocker. I have never adopted this practice but would love to see data that shows it is better in some circumstances. I think the practice does not hurt anything but falls under the heading "just in case." Gayle Callis, among others, may be able to shed some additional light on this.

Happy Friday!

Teri Johnson, HT(ASCP)QIHC
GNF Histology Lab Manager
Genomics Institute of the Novartis Research Foundation
858-332-4752