[Histonet] Two antibodies from the same host with tyramide

[Adam .]

Hi all,

For the past several months, I have been attempting to get double
immunofluorescence with two goat anti-mouse antibodies on mouse FFPE bone.
The antibody that is giving me a lot of trouble is the goat anti-VE-Cadherin
from R&D Systems. Essentially, it seems to work without the tyramide but
when you do the entire procedure, it stops working.

Here is my procedure:

   1. Block peroxide (3% H2O2)
   2. Block with TNB buffer (the blocking buffer that comes with the
   tyramide kit)
   3. Block avidin/biotin
   4. Goat anti-mouse antibody at 1:800. It's undetectable at this titer
   with a directly conjugated secondary.
   5. Biotinylated donkey anti-goat
   6. SA-HRP
   7. Biotinyl tyramide
   8. SA-594
   9. Block with 10 ug / mL irrelevant goat IgG to bind up any leftover
   secondary
   10. goat anti-VE-Cadherin
   11. Anti-goat 649

Unfortunately, I never get any VE-cadherin staining this way. However, if I
do a single stain using that antibody and its secondary, it works great.
I've verified that when staining VE-Cadherin alone, TNB buffer and 10 ug /
mL of goat IgG doesn't alter the staining. Next up are testing peroxidase
block and avidin/biotin blocking, but after that I'll run out of ideas. I've
also tried postponing the SA-HRP and the rest until after all primary and
secondary antibodies are on there, and it still doesn't work.

I'm pretty baffled.

Thanks,
Adam