[Histonet] Re: Methanol in H2O2 explanation

Wed Jul 11 13:22:38 CDT 2012

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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