[Histonet] Re: Two antibodies from the same host with tyramide

[Johnson, Teri]

Adam,

I've been doing some searching and the tyramide shouldn't shelter subsequent IHC reactions, so I don't think that's it. I found evidence of co-localization of mRNA ISH signal with tyramide enhancement and IHC staining without amplification in a publication from J Histochem Cytochem.

I'm actually quite surprised you are getting single staining using the goat anti-mouse antibody from R&D systems. The one I found online (AF1002) has only been tested in Elisa and western blot, and I'm not seeing any sort of antigen retrieval in your protocol. I would suspect that most endothelial markers (with the exception perhaps of Factor VIII) would require it. The fact this is actually working for you in formalin fixed paraffin embedded mouse bone should be cause for celebration.

I still would be interested in knowing if reversing the protocol would have any affect on the staining, especially if you can do the Cadherin first, verify staining briefly, then continue on with the other antibody and its detection and see what happens.

Best wishes,

Teri Johnson