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!!!