I am currently working on optimizing a rapid (1.5 hr) stain for frozen human brain tissue using DAB. My positive slides would show staining, but so would my negative slides. I tried optimizing my negative slide (no primary antibody), but it still resulted with what looked like stained "cells". I decided to run through the protocol without primary or secondary antibody (so the tissue should have been absolutely free of staining), but again, it looked like there were cells that were stained. I have researched artifacts of frozen tissue that might cause this non-specific binding, but have found nothing. Has anyone had this issue with frozen tissue?
Dear Aubrey,
I originally answered your question in another RG thread, but I see that you have also asked it in a question thread.
You were very wise to perform a negative control (no primary or secondary), especially when using DAB.
If you only have applied DAB to your negative control tissue, then you probably have background because you insufficiently inhibited the endogenous peroxidase in the tissue. Hydrogen peroxide can go bad over time, especially the 30%, so make sure you see little bubbles on the tissue when you perform that step. If you do see bubbles, it may be that you have to treat your particular tissue for a longer period of time or with a higher concentration of hydrogen peroxide. When I get this result, I buy new hydrogen peroxide anyway, just to be on the safe side. Only if I still get background in my negative control tissue with new hydrogen peroxide do I start increasing time or concentration.
If instead you amplified your secondary antibody with the ABC reagent (or strepavidin), so that your negative control is ABC + DAB, then it could mean that you have a lot of endogenous biotin in your tissue. You can check this by omitting the ABC reagent, and just applying the DAB (no ABC = no background? Then you do not have a problem with endogenous biotin). If it looks like you have endogenous biotin, then you need to do a special blocking step at the beginning of your protocol to block the endogenous biotin (several companies make a blocking kit).
Good Luck,
Jill
What causes high background in IHC? - ResearchGate. Available from: https://www.researchgate.net/post/What_causes_high_background_in_IHC#view=570bb45d40485432d8555131 [accessed Apr 11, 2016].
This is an image of my stained tissue with no primary or secondary antibody. It is at 20x.
Dear Aubrey,
I originally answered your question in another RG thread, but I see that you have also asked it in a question thread.
You were very wise to perform a negative control (no primary or secondary), especially when using DAB.
If you only have applied DAB to your negative control tissue, then you probably have background because you insufficiently inhibited the endogenous peroxidase in the tissue. Hydrogen peroxide can go bad over time, especially the 30%, so make sure you see little bubbles on the tissue when you perform that step. If you do see bubbles, it may be that you have to treat your particular tissue for a longer period of time or with a higher concentration of hydrogen peroxide. When I get this result, I buy new hydrogen peroxide anyway, just to be on the safe side. Only if I still get background in my negative control tissue with new hydrogen peroxide do I start increasing time or concentration.
If instead you amplified your secondary antibody with the ABC reagent (or strepavidin), so that your negative control is ABC + DAB, then it could mean that you have a lot of endogenous biotin in your tissue. You can check this by omitting the ABC reagent, and just applying the DAB (no ABC = no background? Then you do not have a problem with endogenous biotin). If it looks like you have endogenous biotin, then you need to do a special blocking step at the beginning of your protocol to block the endogenous biotin (several companies make a blocking kit).
Good Luck,
Jill
What causes high background in IHC? - ResearchGate. Available from: https://www.researchgate.net/post/What_causes_high_background_in_IHC#view=570bb45d40485432d8555131 [accessed Apr 11, 2016].
Dear Aubrey,
Blocking is a very important part of immunohistochemical reactions. Blocking of endogenious peroxidase and biotin was already mentioned by Jill.
Other parts which have to be blocked are electrostatic binding sites of proteins. Responsible for these unspecific bindings are in most cases secondary antibodies.
To prevent this you should use either 10% normal sera in the blocking step, later 1% or 1% BSA in TBS/PBS with 0.2 %Triton-X 100. Some labs recommand non fat drymilk as blocking reagent. The choice of normal serum depends of the host of your secondary antibody. For example if you use a goat-anti-rabbit antibody you should use normal goat serum, if you use horse- anti-mouse, then normal horse. The companies from whom you recieve your antbodies sell most the time normal sera as well. The quality of the normal sera is important, becaus bad quality could be responsible for high backgroundstaining.
Another thing is the quality of your DAB- solution. Prepare it freshly short time before you use it. Otherwise it could be oxidized by air and that leads to high background staining as well.
Hopefully this recommands will help you.
Good luck and best regards
Ute
Thank you both for your suggestions. I purchased new hydrogen peroxide and a new staining kit that did not use ABC and my stain looked fairly similar. For the original protocol (with ABC) I used BSA for the blocking step and used serum in the primary antibody incubation. I have tried using 0.2% Triton-X as well.
I have recently been trying to optimize a fluorescent stain and I used 10% serum in my blocking step. I still have not had any luck with it, so I'm thinking I may just have a bad primary antibody.
Dear Aubrey,
If you suspect that you have a bad primary antibody, and you purchased it from a reputable company, their technical services should be able to provide you with troubleshooting suggestions as well as recommendations for control tissue that expresses the antigen of interest. If your lot of antibody won’t label their suggested control tissue with their protocol, ask for a replacement lot.
You may also want to check to see if the primary antibody is in the antibody database at the Journal of Comparative Neurology:
http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291096-9861/homepage/jcn_antibody_database.htm
If it is, that means that someone used it successfully and published the results. I would check that publication to see what the authors used for blocking, etc. There are many different recipes out there, as I am sure you have found.
Good Luck!
Jill
Possible causes of nonspecific staining : highly concentrated primary antibody or prolonged exposure.
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