We are working on staining adult mouse intestines. They are cryo sections. We are getting non-specific binding from the secondary antibodies when no primaries are present. This is supposed to be our negative control.
We have changed channels and we are concerned about anti-mouse secondary being used on adult mouse sections; can that be the source of non-specific binding?
The ultimate goal is to stain these sections for mouse anti histone (h3), rabbit anti caspace, and an in situ TUNEL kit.
Any help would be appreciated. We are at a bit of a loss.
Thanks all!
Tanya,
Yes, using mouse primary antibodies on mouse tissues, like the intestine, can produce high levels of background. The intestine and most other tissues are bathed in fluid from the blood vessels that contain endogenous immunoglobulins, so when you put your anti-mouse secondary onto the tissue it will bind to those endogenous immunoglobulins and produce high levels of background. If you did not fix the tissue by perfusion you are also likely to see very strong non-specific labeling in blood vessels as well.
One way to deal with this problem would be to get a primary antibody against h3 that was raised in a host species other than mouse ( h3 is a popular enough marker that there are probably good anti-h3 antibodies available that were raised in rabbit or goat). This way you would not have to expose your tissue to an anti-mouse secondary antibody.
Another way to deal with this is the use of a "mouse-on-mouse" staining kit, which will provide the reagents and the protocol to essentially bind up all of the endogenous mouse immunoglobulins before you apply your anti-h3. This way only the mouse anti-h3 antibody should be available to the anti-mouse secondary antibody when you apply it.
Good luck,
Dave Sherry
Thank you Dave!
This is very much what we assumed. Of course the mouse antibody was all that was on hand and the person running the project did not want to wait to order a new stain. I really appreciate this answer.
I've found some protocols for binding up the endogenous igs so that may be another way to address this issue.
Thank you again!
Tanya
You said that secondary alone was your negative control. Have you tried the no antibody control? Tissues can have a fair amount of autofluorescence. Before taking steps to change your secondary, I would make sure it's not background autofluorescence. Good luck.
You have to block very well before adding the primary antibody and that involves additional steps. I have used a commercially available kit for that, the Mouse on Mouse Basic KIt from Vector Labs, that has the mouse Ig blocking reagent and it works pretty well. However, you need to have titrated your primary antibody first so as to reduce any non specific binding. Fast, easy and good results. Hope this helps!
Thank you Michael and Christina.
Michael, I very much agree with what you said. I would like to see a control with no antibodies as well, however it's not my project, I am just helping out at the moment. Thought I think that would give us some valuable information.
Christina, thank you. This may be something we will also try!
Tanya
As many has mentioned here, M.O.M blocking kit is one options. Another option is to use Rodent Block from Biocare Medicals. Incubate for 45 minutes before primary. Hope it helps
Hi Tanya,
The best mouse-on-mouse kit in our hands is the Animal Research Kit from Dako. The system utilizes streptavidin-biotin binding, so if endogenous biotin is not a problem you can give it a shot.
Cheers,
Ivan
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