Fluorescent immunohistochemistry
Mice were deeply anesthetized and perfused transcardially with 20 ml of
cold heparinized saline followed by 20 ml of 4% paraformaldehyde.
Brains were removed and immersed in paraformaldehyde and then dehydrated
with 30% sucrose for 72h. 30 µm thick sections were cut on a freezing
microtome. Free-floating sections were rinsed twice in
phosphate-buffered saline (PBS) and twice in PBS with 0.2% Triton
X-100. Sections were blocked by incubation in PBS with 1% bovine serum
albumin (V900933-100G, Sigma–Aldrich, USA) and 0.2% Triton X-100 for
45min at room temperature. Then incubated with primary rabbit
anti–c-Fos (1:400; 2250S, Cell Signaling Technology, USA) for 24h.
After washed three times, the sections were incubated with a secondary
antibody Alexa Fluor 488 donkey anti-rabbit (1:500; Invitrogen, USA) at
room temperature for 2h. The samples were then rinsed in PBS for 10min;
confocal images were taken with a confocal fluorescent microscope
Pannoramic MIDIII (3D Histech, Hungary) and further processed using the
software CaseViewer.