General Research Topics > Neuroscience

Mouse brain IHC - free floating method - help with extravidin-DAB!!!

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LRenee:
I'm hoping someone can help me with this. I am attempting to do IHC on free floating brain sections in mouse (40um thick). I am using a protocol provided by the first author of a publication that got beautiful results. I am also using all of the same primary and secondary antibodies (secondary is biotinylated - sigma), extravidin (sigma), and DAB kit (vector), along with the same dilutions as the publication. I am getting strong staining of the entire section within 30 seconds in the DAB solution, I have contacted the author about this and they can't seem to help me. Here are my controls:

1) tissue section only - this had no primary, secondary, or extravidin to test for endogenous peroxidase activity (which I block with H202) - I got NO staining - YAY.
2) no primary - this had secondary and extravidin - I got lots of background. Sad. I know it's not non-specific binding of primary.
3) no secondary - this had primary and extravidin - funny thing, tons of background, and it's my secondary that is biotinylated, so I know it's not non-specific binding of secondary.

This makes me think that somehow extravidin is binding my tissue non-specifically? Has anyone seen this? I know that there are biotin/avidin blocks, but this wasn't done by the author of the publication, and according to some other posts on this forum, this may actually increase background?

Thanks for any suggestions!!!

MDeng:
Try all tests including all control on the frozen section on slide, if there is not background shown on the control sections, it means washing may be the problem. If same problem happen as with floating section, try Avidin/Biotin block kit.

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