Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Immunofluorescence for beta catenin and VE-cadherin


  • Please log in to reply
1 reply to this topic

#1 haiyan

haiyan

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 11 March 2009 - 12:58 AM

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.

Thanks

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,826 posts
413
Excellent

Posted 11 March 2009 - 03:32 PM

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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.