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.