I have had the same problem stated as Koi, I would like to explain mine..I am looking to map the C-fos in the V1 of the rat brain.
1. I use Brain tissue slices, PFA perfused, Fixed, sectioned and mounted on slides and frozen until further use.
2. Used TBS-twn-20 for washing steps.
3. Nonspecific blocking for 2hrs by serum (10%).
4. Primary antibody I used first was from Santa Cruz Dilution 1:100 overnight incubation
5. Secondary antibody Alexa Fluor-594 dilution 1:300 incubation for 2 hrs at room temperature
6. Washed several times and add the DAPI incubated for 20 min
7. Washed, added the antifade and cover slipped.
the problem is I am seeing positive neurons (mean the antibody is binding to neurons which can be seen in the control tissue, which is from the rat not exposed to any visual stimulation [that's what i do to induce the c-fos in the V1 area f the brain ])
I changed the antibody from santa cruz to ABcam antibody, however I am still seeing the same result :-(
can any one help me in solving this problem.