6 replies to this topic

[wdyeo]

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.

[bob1]

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.

[wdyeo]

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 .

[bob1]

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.

[wdyeo]

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.

[bob1]

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.

[wdyeo]

HI,
OK, Thanks alot for the suggestion