AW: [Histonet] RE: Re: Methanol in H2O2 explanation

[Gudrun Lang]

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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