Kim, The direct IPX is the easiest method apart from a direct immunofluorescence. Very few steps: 1. Block endogenous enzyme 2. Block non-specific Ab binding 3. Put the HRP-conjugated Ab on (a an appropriate dilution, usually more concentrated than if you were using an ABC or Polymer amplification method) 4. Incubate in your usual DAB-H2O2 solution 5. Counterstain and you done. Throw in a few buffer washes in between and maybe some antigen retrieval if required. We use the direct IPX method for renal biopsies (IgG,M,A etc). See Birchall, I.W., (1996) "Direct antibody method for formalin fixed, paraffin embedded renal biopsies: a comparison with the peroxidase labelled streptavidin/biotin method" J. Histotechnol 19(2): 125-129.
Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) 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: ih...@googlegroups.com [mailto:ih...@googlegroups.com] On Behalf Of Kim Merriam Sent: Thursday, 21 May 2009 10:42 PM To: Histonet; ih...@googlegroups.com Subject: [IHCRG] HRP-labeled primary antibodies Hi All, Has anyone had experience doing IHC on FFPE tissues with HRP-labeled primary antibodies? I was wondering what the best way to detect them would be. I assume that going strait to DAB would not work, since no amplification is there. I was thinking of using a biotinyl tyramide step to amplify the signal. Also, do you think the final antibody concentration would need to be higher than with traditional, unlabeled primaries? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ********************************************************************* 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