[Histonet] Re: Methanol in H2O2 explanation

[Edwards, Richard E.]

We use hydrogen peroxide in a sodium  azide solution,(on acetone fixed GMA processed tissue) works fine.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of McMahon, Loralee A
Sent: 13 July 2012 15:18
To: Rene J Buesa; Hobbs, Carl; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Re: Methanol in H2O2 explanation

I think that the original question was why is it recommended in some procedures that H202 be diluted in methanol.  And others say distilled water or buffer.  What makes one better than the other?
Some people I have worked with insist on getting 30% h202 and diluting it in methanol to a 3% solution.   Others use 3% that you buy from the drug store.  No one seems to know why except that is how it has always been done.  

Loralee McMahon, HTL (ASCP)
Immunohistochemistry Supervisor
Strong Memorial Hospital
Department of Surgical Pathology
(585) 275-7210
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa [rjbuesa <@t> yahoo.com]
Sent: Friday, July 13, 2012 10:07 AM
To: Hobbs, Carl; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Re: Methanol in H2O2 explanation

I have been fascinated by this long discussion about H2O2 and its use in IHC procedures.
We have to start from the fact that ALL cells contain peroxidase because ALL cells respire and ALL cells need to use oxygen they receive from blood (or hemolymph in crustaceans and other arthropods).
The initial IHC procedure were peroxidase-anti-peroxidase (PAP) methods (and many remain as such).
A fixed cell = a dead cell BUT in spite of being dead, the peroxidase they contain is still able of reacting with the PAP complex.
The cell, being dead, cannot make more peroxidase, but there is still peroxidase in it after any type of fixation, even with NBF, as Carl points out.
IF that peroxidase is not "quenched" = INHIBITED the use of a PAP IHC procedure on that uninhibited peroxidase = excessive background in the section after the IHC method is completed.
So, how to avoid that background? By "quenching" or inhibiting the peroxidase contained in the cell.
How? With a substance rich in oxygen that will inactivate the peroxidase by "spending" it and that substance is H2O2 used at the start of the IHC protocol.
René J.


________________________________
From: "Hobbs, Carl" <carl.hobbs <@t> kcl.ac.uk>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, July 12, 2012 3:27 PM
Subject: [Histonet] Re: Methanol in H2O2 explanation

"H202 mixed with Buffer here.(instead of aq.) That's OK too, right?"

Right.

An addition to the misconceptions that Tim interestingly elucidates: some use H2O2 in buffer because they think that , as the enzyme is performing a physiologic catalytic reaction, it should be at a physiological pH, in a physiological buffer.
Well....if it's a frozen section, it's been fixed; if it's a P.wax section, it's been fixed in Formalin, fixed in alcohol, subjected to 60C heat.....so, the fact that endog. Peroxidases survive those assaults, indicates that physiologic conditions no longer apply?

Re use of methanol/ethanol....I use IMS. Always have done.
NB: great excess of substrate can " kill " enzymes ( irreversible substrate inhibition): that is why we add a great excess of H2O2 ( substrate) to "kill" endog. Pxs.
We then use a stoichiometric quantity of the same reagent ( H2O2) in the presence of DAB/AEC, to visualize  sites of AB:Ag interaction.

"For the enzymatic activity of peroxidase it needs an electron-donator"
The enzymatic activity of peroxidase needs no electron donor, as regards Immuno.....it will "digest" H2O2 into water and oxygen gas.
However, if you wish to use HRP as a reporter molecule, you must also add a suitable chromogen ( DAB, AEC) that


Carl Hobbs
Histology 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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