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

restain secondary antibody


  • Please log in to reply
6 replies to this topic

#1 wdyeo

wdyeo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 54 posts
4
Neutral

Posted 02 May 2013 - 12:37 PM

Dear all,

I am doing the immunohistochemistry on the tissues, in the gel about 150um (cut with vibratome).
I made a mistake in my secondary antibody, instead of using secondary anti-rabbit green + secondary anti-mouse red; i used secondary anti-rabbit red and secondary anti-mouse green . I did a double staining for my primary antibodies, one is against rabbit and the other is mouse.

Is there a way to remove the secondary antibody and restain my tissues slice?
Thank you.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,831 posts
415
Excellent

Posted 02 May 2013 - 07:59 PM

So what's the problem? Sure, it isn't consistent with all your other samples, but there isn't a real issue with this other than you having to note that the sections were stained opposite to what you usually do.

#3 wdyeo

wdyeo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 54 posts
4
Neutral

Posted 03 May 2013 - 06:18 AM

Hi,
The problem i am facing now is, i try to identify GFP staining in my tissues and one of the primary antibody is against the GFP and i am supposed to stain the GFP with secondary in green to enhance the GFP signalling but i made a silly mistake, using red for the GFP antibody Posted Image .

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,831 posts
415
Excellent

Posted 04 May 2013 - 01:08 AM

OK. It is difficult to dissociate antibodies from their specific targets so any stripping protocols will also remove primary staining and are very likely to damage the tissue/antigens themselves. Your best bet is to repeat the slides.

#5 wdyeo

wdyeo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 54 posts
4
Neutral

Posted 09 May 2013 - 12:24 AM

Yeah....but i do not have the sample already :<
May i know what is the commonly used stripping protocols...at least there's still some slight hope to try...
Thank you.

#6 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,831 posts
415
Excellent

Posted 09 May 2013 - 12:49 AM

I don't know of any stripping protocols for IHC/IF, though 0.1 M glycine at low pH 2-3 for a minimal time might work. The problem will be damage to the tissue and/or antigens, so you will have to repeat it anyway to be certain, even if you do manage to find a protocol that works.

#7 wdyeo

wdyeo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 54 posts
4
Neutral

Posted 10 May 2013 - 12:58 AM

HI,
OK, Thanks alot for the suggestion :D




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

©1999-2013 Protocol Online, All rights reserved.