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.ho...@kcl.ac.uk> To: "histonet@lists.utsouthwestern.edu" <histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
