[Histonet] Re: Methanol in H2O2 explanation

Wed Jul 11 18:06:15 CDT 2012

So, the thinking at the time was to kill the endogenous peroxidase twice!

No one can say that us oldies weren't thorough!

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gudrun Lang
Sent: Thursday, 12 July 2012 4:23 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: Methanol in H2O2 explanation

Ha! I've found it. It can be found in Dr. Kiernan's book in the chapter enzymehistochemistry - the thing, that an electron-donator is used for demonstration.
 And he states, that methanol alone also inhibits peroxidase - perhaps by the fixation = denaturation effect on the protein?

In the following publication one can see the equations for the peroxidase-reaction and this phrase: 
"In this study, we have used a steady state kinetics approach to demonstrate that MPO is irreversibly inactivated by excess H2O2 in the absence of reducing substrate."
The Open Enzyme Inhibition Journal, 2009, 2, 28-35 Mechanism-Based Inhibition of Myeloperoxidase by Hydrogen Peroxide: Enhancement of Inactivation Rate by Organic Donor Substrates

It's rather exhausting to prove the own statements  ;-) puh.

Gudrun

-----Ursprüngliche Nachricht-----
Von: Morken, Timothy [mailto:Timothy.Morken <@t> ucsfmedctr.org]
Gesendet: Mittwoch, 11. Juli 2012 18:12
An: gu.lang <@t> gmx.at; histonet <@t> lists.utsouthwestern.edu
Betreff: RE: [Histonet] RE: Re: Methanol in H2O2 explanation

It's funny how histology lore gets passed down, passed around and misunderstood over the decades. When I ask questions in labs about why they do a certain procedure they can rarely name a source (besides a particular long-retired particular tech who wrote the original procedure) or give reason as to why it is done that way. Even if it works well, at least people could take the time to understand why!

When I was researching buffer compositions long ago I found a dozen variations of PBS alone (and only a dozen because I just stopped looking).
When you delve into these things it becomes clear that people made their own solutions for a particular purpose. Maybe they wrote a paper, taught a course or wrote a book. Then that formula was copied by many others and their particular formulation became the "state of the art" even if the "art"
practiced by someone else had little to do with the original pursuit (the Sheehan book is full of variations of stains that were originally for specific research purposes. There is precious little guidance in any books about what is good for a particular use!).

The reason for using methanol / H2O2  that I was told long ago was prevent frozen sections from being damaged by H2O2 bubbling. In fact, we would fix frozens in cold acetone and THEN put the slides in cold methanol/H2O2 (double fixation?)  In that lab we never used methanol for deparaffinized sections. Since I had no exposure to the clin lab I never knew about using methanol for blood smear fixation. From literature searches it seems methanol, rather than ethanol, is used primarily because it is cheaper. In other words, it does not appear to be a magical substance!

The questions asked here are good ones. However, hopefully those reading and responsible for producing procedure manuals will include the reasoning for each step of the method and not just assume it is necessary, or "common knowledge," just because it has been handed down through generations of long suffering techs!

Tim Morken

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gudrun Lang
Sent: Wednesday, July 11, 2012 7:17 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: AW: [Histonet] RE: Re: Methanol in H2O2 explanation

For the enzymatic activity of peroxidase it needs an electron-donator (or receptor - I can't find the literature...) in the vicinity; therefore H2O2 and DAB are added ad once, and DAB is oxidized and transformed into the insoluble, amorphe substance through polymerization.
Without the donator H2O2 in excess works as inhibitor and blocks the activation-side of the enzyme.
I think H2O2 in methanol was primarly preferred, because the frozen slides were fixed at the same time.

Rehydration after dewaxing depends on the following reagens.

Gudrun

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Tony Henwood (SCHN)
Gesendet: Mittwoch, 11. Juli 2012 06:21
An: 'Hobbs, Carl'; histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] RE: Re: Methanol in H2O2 explanation

Hi Carl,

What do you mean by "Why do people rehydrate after dewaxing?" Do you really mean that slides do not require rehydration or do you mean that slides can be left to dry after de-waxing prior to staining.

Re-hydration is necessary, otherwise xylene will prevent aqueous stains from doing their thing efficiently.

I was lead to believe that as H2O2 was catalysed by endogenous peroxidase, the reactive oxygen reacted with the methanol to then degrade the enzyme, but I need to look closer at this chemistry.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Hobbs, Carl
Sent: Wednesday, 11 July 2012 1:32 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: Methanol in H2O2 explanation

Why do some people use methanolic H2O2?
Why do people rehydrate after dewaxing?
Both are unnecessary, under usual conditions.

Methanol was used in the early days as a peroxidase blocker by itself.
The combination was devised as a "belt and bracer" method.
As you stated, you use aq H2O2 effectively.
So do I and many others.

However, for unfixed frozen sections, I would never use aq H2O2, if I wanted sections remaining on my slides....

After dewaxing, rinse sections in 4x 100% alcohol, rinse in water, endog. Px block while you make up you AR solutions....

Carl Hobbs
Histology and Imaging Manager
Wolfson CARD
School of Biomedical Sciences
Kings College London
Guys Campus
SE1 1UL
Tel: 020 78486813
Fax: 020 78486816
020 78486813

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