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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
