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Author Topic: How to reduce non specific staining of your primary antibody?  (Read 19304 times)

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Offline funk.106

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Re: How to reduce non specific staining of your primary antibody?
« Reply #15 on: February 18, 2011, 10:31:33 AM »
Regarding PBS versus TBS, generally TBS is used for western blotting and PBS is used for IHC, but I doubt that it really makes a difference. I do think that tween will improve washes though (see post from Feb 4th).
As far as blocking agent only binding non-specific binding, no I don't think it only recognizes the non specific sites. More so, I think that the specific binding of the antibody is stronger than to non specific, and so it more readily binds the sites regardless of whether there is blocking agent in the way, and doesn't as much to the non-specific sites that are blocked.

Re: How to reduce non specific staining of your primary antibody?
« Reply #15 on: February 18, 2011, 10:31:33 AM »

Offline JB22

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Re: How to reduce non specific staining of your primary antibody?
« Reply #16 on: February 21, 2011, 05:14:09 AM »
I am assuming ABC is an avidin-biotin amplification.  If so, I am not seeing a blocking step for endogenous biotin.  This could be causing your background issues....




Yes ABS is an avidin-biotin amplification. No I do not block endogenous biotin. I didn't know that tissue can have endogenous biotin. I asked around to get some more information and nobody at my lab blocks endogenous biotin.

But I do not think that this would be the reason for my background sins I perform staining with different antibody's on the same tissue that do not have any background. Also with the tissue sections were I do not use primary antibody I have no staining.

I tried several concentrations of secondary antibody. This does reduce the intensity of the staining but doesn't seem to prevent the background for the concentrations I tried, 1/200, 1/500, 1/1000, maybe I need to go even lower.

Another thing I tried is to not perform antigen retrieval. This seems to do the trick, I do not know why but if I do not perform antigen retrieval my background is almost gone. I am going to test another batch of slides to see if I can repeat this result.

« Last Edit: February 21, 2011, 05:19:32 AM by JB22 »

Re: How to reduce non specific staining of your primary antibody?
« Reply #16 on: February 21, 2011, 05:14:09 AM »