C fos 9f6 rabbit mab

60 min after optogenetic stimulation, mice were perfused with PBS followed by 4% paraformaldehyde (PFA) (cat. no. P1110, Solarbio) in PBS. Brain slices 10 µm thick were dissected in paraffin. Slices were then blocked by using 10% normal goat serum + 0.3% Triton in PBS +5% BSA for 2 h at room temperature. Slices were then placed in primary antibody solution (c‐Fos (9F6) Rabbit mAb, Cell Signaling Technology) diluted (1:500) in blocking buffer at 4 °C for 16–18 h. After washes with PBS, slices were incubated with secondary antibody (Goat anti‐Rabbit IgG (H+L) Cross‐Adsorbed Secondary Antibody, Alexa Fluor 488, Invitrogen) diluted (1:1000) in the blocking buffer for 2 h at room temperature, followed by washes with PBS. Slices were allowed to dry and mounted on glass slides with Prolong Diamond Antifade Mountant with DAPI, capped with a coverslip and then imaged. Samples were imaged by using an upright confocal microscope, in the UBSN facilities of The Hong Kong Polytechnic University. Cells showing both c‐Fos and mCherry signals were counted manually in five FOVs in each brain slice, and the percentage of c‐Fos⁺ cells was counted per slice imaged.