I recently tried an ERa immunofluorescent stain using strep488 as the secondary. There are clearly ERa positive cells, but I'm not sure what these "dots"/noise are in the background of my GFP channel.
Skylar King, that's what I thought too, but seeing "dots" as non-specific binding is new to me. The ERa primary was on for 48 hours. Slides were then rinsed with PBS three times for 5 mins (15 mins) before being incubated in secondary for two hours. After that, 2x5 min PBS washes, dapi for 5 mins, then a final 5 minute PBS rise. Slides were then dried overnight in the dark and coverslipped the next day.
Try doing 3 two minutes washes after the secondary antibody incubation. I find that the shorter washes helps with the nonspecific. Do you see the nonspecific binding on every single slide? Are the bright spots also positive for your DAPI staining?
I've talked to a few people about this now and I'm realizing that I may not have been clear about what "dots" I'm talking about. The bright spots are definitely artifact or non-specific binding. What I'm talking about is circled in the image I reuploaded.
Are you blocking before your primary antibody incubation? That does not really look like noise to me, it is definitely binding to some sort of structure and they are only present in the GFP channel?
Skylar King, yup! One hour in blocking at room temp and I only see this in the GFP channel. I don't know if this matters, but the tissue is freshly frozen and fixed with 4% PFA on the slide after mounting and before blocking.
I agree that it looks like it's binding to something. I see a lot of it in other sections, so I'm not confident it's all ERa.
I would try using a different secondary to see if you still see the dots in the TRITC channel. Sometimes we do get nonspecific binding in the tissue but it does not look as specific as this, it is just super bright and it is outside of the artery. Have you looked to see if your tissue has any autofluorescence?
Skylar King, I looked back at my tissue. All those "dots" are really cells. This was my first time staining for receptors. I didn't realize just how abundant ERa is in the brain! Thank you for your help.
Check that the signal is not blead through from DAPI. We see this sort of problem when very high levels of DAPI are used. Stain a sample without the DAPI staining to be certain!