I'm trying to stain a membrane protein in frozen sections of brain tissue. Histology is new to me, but there are members of our lab whom are wizards when it comes to muscle tissue. I've run into issue with high background, and I can't figure out why. Here is the protocol:
Tissue Extraction and Processing
1) Brain tissue was removed, and fixed in 4% paraformaldehyde-PBS. Snap frozen in OCT, stored at -80C.
2) Sectioned tissue at 10um. Placed on slides, stored overnight at -20C
1) Removed slides from freezer. Allowed to warm at room temperature for 15 minutes.
2) Washed in PBS 3 times, 5 minutes each
3) Permeabilized with 1% Triton-X-PBS, 20minutes at room temperature
4) Washed in PBS 3 times, 5 minutes each
5) Incubated with 10% Normal Goat Serum, 15 minutes at room temperature
6) Primary antibody, rabbit polyclonal; 1:50, 1:25 (tried both dilutions separately); 60 min @ 37 C
7) Washed in PBS 3 times, 5 minutes each
8) Incubated with 10% Normal Goat Serum, 15 minutes at room temperature
9) Secondary antibody. Goat anti-rabbit IgG-FITC. Tried 1:300, 1:500, and 1:1000. 30 min at 37C
10) Washed in PBS 3 times, 5 minutes each
11) Added DAPI, cover slide, and viewed.
My negative controls (processed the same as above, except PBS-BSA was added in place of Primary antibody), look the same as my samples. There is a lot of background, with the occasional brighter green. The brighter green does seem to be localized to cells, which is extra confusing.
When there isn't a lot of signal, will the microscope increase its "signal" to try to find the "positive" areas? So if there wasn't any FITC on my samples or negative control, then you would expect to see a lot of green background? I tried adjusting the exposure, but I can't seem to get rid of the green background on my samples or negative controls.
Anyone have any idea what's going on? Is this a microscope issue, and both my slides are just negative for the protein? My lab mate gets nice muscle images, free of background, but he's having trouble communicating (language barrier).