I am doing immunofluorescence(double staining) on tissue section (Tonsil tissue). I tried it for 3 times. But each time I find lots of backround in the isotype control. I have improved my washing steps (by replacing the washing solution (0.1% BSA in PBS) for every wash). The blocking method I used is to incubate the slides in 3% oxidising hydrogen peroxide in absolute methanol for 10 minutes. I have not tried using any serum for blocking.
In my test samples, I found some staining. But when I compared the stained spots with the DAPI stained image, I found most of the stained spots were from dimly stained DAPI region. Brightly DAPI stained regions dint have any stain. Does it mean that my staining is unspecific?