[Histonet] EGFP IHC in floating brain sections

[Caroline Bass]

Hello,

I¹m hoping someone could help me troubleshoot my staining protocol. I am
staining to EGFP in microglia (produced by a virus I injected in the brain).
Here¹s my general protocol:

Deactive endogenous peroxidases (0.1M PB + 20% methanol + titon-x100 (80 ul
in 40 ml total) + 0.3% h2O2) for 15 min.
Wash 3X15min in 0.1MPB+0.3%titon
Incubate in 0.1MPB+0.3%triton+1%normal donkey serum for 1 hr
Incubate with primary (goat anti-GFP from Rockland) overnight at 4 degrees
(1:1000)
Wash 3x15 min 0.1M PB + 0.3% triton
Incubate in with secondary (Jackson Donkey anti-goat, biotin labeled)
overnight at 4 degrees (1:500)
Wash 3x15 min in 0.1M PB

Use ABC kit and vector SG for substrate (can¹t remember how long I let it
develop, but it was within a couple of minutes).

So, the bottom line is that I did a test section and I thought it had a lot
of background, so I incubated the other sections for less time. However,
when I took a closer look, the sections with little background had no
microglial signal, while the one with high background had a very clear and
noticeable signal in the place it should be. So, the question is how to cut
back on the noise. Would increasing the secondary concentration to 1:1000
help? I¹m following a protocol someone else came up with, but it seems
strange that the secondary is at a higher concentration than the primary.

Thanks!

Caroline