I am investigating VE-Cadherin expression in Human Coronary Artery Endothelial Cells towards certain compounds. VE-Cadherin is a membrane protein. I got (attached picture) VE-Cadherin expression even in a plasma membrane. I am wondering if that occurred as a result of permeabilisation step in immunocytochemistry or as a result of high Antibody concentration?
Thanks in advance for any comment.
Hi,
Whether you need a permeabilisation step for this antibody depends on which epitope the antibody recognises, i.e. does it recognise an epitope located in the external part of the VE-cadherin molecule, or an epitope on its cell-internal part?
Your picture also looks as though you might have quite a bit of non-specific or background staining. You could try and use a lower antibody concentration, amongst other possibilities.
Hey Christine,
Thank you very much for your response. Theoretically this antibody should recognise external domain, although the company recommended the permeabilisation step. My concern is about high background staining that you already noticed, I may first go with a lower antibody concentration.
Appreciate your comment an thoughts, it is really helpful.
Thank you.
Looks like you have a lot of background staining. It is probably an antibody concentration or blocking problem you have here. Maybe its even the antibody you are using. In this paper you can find a protocol and an antibody that works really well for VE-cadherin stainings: http://www.ncbi.nlm.nih.gov/pubmed/24166953
If you send me your protocol I could check it if you want.
Thank you very much Anouk, I do appreciate your help. I reduced the primary AB concentration and still have the same problem.
What do you use to block? Also try reducing the secondary Ab concentration.
I am doing the assay before getting 100% confluent cells, does that make any difference?
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