I am getting very specific staining using my antibodies; however, I also get (sometimes) positive staining around the edges of my preparation (tissue sections, FFPE). I have tried both for antigen retrieval, heat and protease. Can anyone explain why this happens and how to avoid it? any reference I can check at?
Thanks in advance.
I don't really know what causes the edge effect, though I suspect that it is due to a peripheral shift of the antibody concentration during incubation. What I do know is that I rarely see it to a significant degree anymore, with the staining parameters that I currently use. I do all staining on an automated stainer, using 300 ul volumes of all solutions, with a PAP pen marking around the section. All our incubations are 1-2 h at RT only, I don't do any overnight incubations. With these parameters I don't really see edge effect.
It could also be your secondary antibody sticking to the edges. Using fresh secondary at a lower concentration and doing a couple extra washes may help. Also, a secondary only negative control for comparison.
1. Use a wet chamber to prevent drying during incubation. Use enough staining solution to cover whole slide and a bit of the surrounding.
2. Wash thoroughly (!) between all staining steps.
3. If possible without loss of staining solution: Incubate with soft agitation (lab rocker).
Generally: Don't forget proper control incubations.
It might be too late to add the answer but Abcam mentions that under-fixation can lead to edge staining.
http://www.abcam.com/protocols/ihc-fixation-protocol
I'm having a similar problem. but I don't think it was because of the lack of fixation. because it is more likely to see a lack of fixation in the middle of the tissue than in the corners. I usually try to interpret nonspecific binding according to staining in negative samples.
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