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Immunofluorescence for beta catenin and VE-cadherin - to see the colocalization (Mar/11/2009 )

Hi, I want to detect the colocalization of beta catenin and VE-cadherin in same sample of HUVEC. The primary antibody for beta catenin is from rabbit and the primary antibody for VE-cadherin is from mouse. The senconday antibody for beta catenin is Alexa 488 goat anti-rabbit and senconday antibody for VE-cadherin is Alexa 568 rabbit anti-mouse.

I added the two primary antibodies at the same time to a same sample and incubated for 1h at 37 degree. After that, I added the senconday antibody for beta catenin (Alexa 488 goat anti-rabbit) first and incubate for 1h at 37 degree. After that, I wash the sample 3-4 times with PBS and then add the senconday antibody for VE-cadherin (Alexa 568 rabbit anti-mouse) and incubate.

The problem is I cound not see much beta catenin, which is quite different from the results before when I only stained beta-catenin. The Ve-cadherin is not strong as well.

Is there any problem in my procedures? Please let me know your advices.



Titrate the antibodies: Do a dilution series for each primary antibody and us a fixed dilution of secondary (1:1000 is good for alexafluor)