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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Tony Henwood (SCHN) Gesendet: Mittwoch, 11. Juli 2012 06:21 An: 'Hobbs, Carl'; histonet@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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Hobbs, Carl Sent: Wednesday, 11 July 2012 1:32 PM To: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet **************************************************************************** ***** This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. **************************************************************************** ***** _______________________________________________ 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