We have been trying to visualize endothelial cells in heart sections, but it has failed every time. We have tried two different Vegf primaries and a Vegf R2 primary that a colleague has used successfully many times before. Our secondary antibodies (Alexa Fluor 594) work with other primaries (vimentin and Adamts2), but do not fluoresce with any of our EC primaries. We tried the Invitrogen Tyramide Boost Kits, both the streptavidin and HRO versions. For the EC slides the tyramide kits were entirely unspecific, but with our vimentin primary, they worked fine.
We have tested all of our secondary antibodies, the boost kits, and our protocol, and they all work on our cardiomyocyte and fibroblast markers, and specific gene primaries. It's only when we use our endothelial markers that they fail.
Any thoughts on what is going wrong or what we could change?
Are you doing an antigen retrieval step on your sections? Could it be that isn't working? There could also be batch variability in your VEGF primary even if your colleague previously got it to work. Probably worth going back to the company to see what they say. Can you try a different secondary with an alternative fluorophore? Could be some crosstalk if you are doing this in multiplex. Hope you get it to work!
A few questions.....FFPE or cryo sections? Fixation? Can you give us a quick summary of your protocol?
Sections are 5um cryo sections. Steps are fixation - 5min 4% formalin, antigen retrieval - three five minutes washes with PBST, permeabilization - 10 minutes in 0.1% Triton, blocking - 1 hour in serum free block, primary antibody overnight at 4C, wash 3 times with PBST, secondary antibody for 1 hour, dapi, image.
I know our 594 secondary works, but I have not tried an alternate fluorophore. We have a few options to try there.
If you know your secondary works then it's looking like a problem with detecting your antigen. Your fixation is fairly standard but have you tried anything else like methanol or acetone? What retrieval steps are you using? If it's heat then maybe you need to try an enzyme instead such tryspin or pepsin (although these can damage antigens!). Have you got a control sample expressing your antigen such as a cell line etc that you can check your primary works on? Would give you confidence in the antibody and you could narrow it down to fixation/retrieval. Not sure if that helps!
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