[Histonet] Re: Methanol in H2O2 explanation

[Hobbs, Carl]

"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