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

[Johnson, Teri]

Adam,

Thanks for the detailed protocol. I can't explain it either but I'll throw some ideas out there and maybe it'll stimulate discussion. Hopefully Chris vanderLoos will chime in.

Have you tried doing the labeling in reverse, using the VE-Cadherin first and then blocking with the goat IgG, and then doing the other antibody/tyramide protocol? I'm not sure what effect, if any, the hydrogen peroxide might have on the anti-goat fluorescent 649 label, so you could certainly do that block prior to doing the first primary antibody incubation.

Also, you may have to double the concentration of the Cadherin to get it to label at the same intensity as you do in single staining. But still I might have expected to see a signal.

Do the two antibodies co-express? Could there be something with the covalent binding of the HRP-tyramide complex that shelters the antigenic sites for the second antibody? That's why I wondered what might happen if you reversed your protocol.

Let us know if you get it figured out and what fixed it for you.

Good luck,

Teri Johnson, HT(ASCP)QIHC
Managing Director, Histology Facility
Stowers Institute for Medical Research
Kansas City, MO