Mice were transcardially perfused with PBS followed by formalin. Brains
were post-fixed overnight in formalin and placed in 20% sucrose for 24
h. Free-floating sections (40 µm) were prepared with a cryostat, blocked
(3% BSA, 2% NGS, and 0.1% Triton-X in PBS for 2 h), then
incubated with primary antibody (chicken anti-GFP, Abcam, ab13970, 1:1000; rat
anti-RFP, ChromoTek, 5f8, 1:2000; rabbit anti-cFos, Santa Cruz Biotech, sc52,
1:1000) overnight at 4°C (two nights for cFos staining). Sections were
then washed, incubated with secondary antibody (Alexa Fluor 488 goat
anti-chicken, Life Technologies, a11039, 1:1000; Alexa Fluor 568 goat anti-rat,
Life Technologies, a11077, 1:1000; Alexa Fluor 568 goat anti-rabbit, Life
Technologies, a11011, 1:1000) for 2 h at room temperature, mounted, and imaged
with a Zeiss LSM 700 confocal microscope. Sections stained for cFos underwent
unmasking before blocking (1% H2O2 + 1%
NaOH in PBS for 10 min; 0.3% glycine in PBS for 10 min; 0.03%
SDS in PBS for 10 min).
were post-fixed overnight in formalin and placed in 20% sucrose for 24
h. Free-floating sections (40 µm) were prepared with a cryostat, blocked
(3% BSA, 2% NGS, and 0.1% Triton-X in PBS for 2 h), then
incubated with primary antibody (chicken anti-GFP, Abcam, ab13970, 1:1000; rat
anti-RFP, ChromoTek, 5f8, 1:2000; rabbit anti-cFos, Santa Cruz Biotech, sc52,
1:1000) overnight at 4°C (two nights for cFos staining). Sections were
then washed, incubated with secondary antibody (Alexa Fluor 488 goat
anti-chicken, Life Technologies, a11039, 1:1000; Alexa Fluor 568 goat anti-rat,
Life Technologies, a11077, 1:1000; Alexa Fluor 568 goat anti-rabbit, Life
Technologies, a11011, 1:1000) for 2 h at room temperature, mounted, and imaged
with a Zeiss LSM 700 confocal microscope. Sections stained for cFos underwent
unmasking before blocking (1% H2O2 + 1%
NaOH in PBS for 10 min; 0.3% glycine in PBS for 10 min; 0.03%
SDS in PBS for 10 min).