[Histonet] problem with double IF of brain section

[Bass, Caroline]

Hello everyone,

I’m trying a double stain for GFP and tyrosine hydroxylase in floating brain sections. Rat, perfused with formalin. I’m having a big problem, in that it’s clear that the top side of the sections are well stained, as are the bottom, but everything in between is not. These sections are 50 microns. Any suggestions? I assume it’s a penetration problem. But I’m not very comfortable with IF in general.

Here’s what my tech does:

Placed sections each in a well of a 24 well dish  and rinsed in PBS (to remove the Na azide), 1 X 5 min. Rinsed in PBS-0.5% Triton X-100, 3 X 10 min. Incubated tissue in PBST (0.25% Triton X) + 5% NGS, 1 X 60 to 120 min. Incubated sections in 1ary antibody in PBS-T(0.25%) overnight @40C (covered to minimized evaporation). Both primaries together.
Tyrosine Hydroxylase  1: 4,000 ImmunoStar #22941 (mouse)
a- GFP  1:2,000  Invitrogen 6455 (rabbit)
Next day, rinsed tissue w/ PBS-T, 3 x 10 min. Incubated w/ 2ary ab : Alexa 555 donkey anti mouse 1:4,000, Alexa  488 goat anti rabbit, 1:2,000, 1 x 2hs @RT. 2ary ab diluted in PBS. Rinsed w/ PBS, 3 x10 min. Mounted on slides, coverslipped using ProLOng Gold w/ DAPI. Cured in dark place @ RT overnight.

Thanks,
Caroline