1950 cm cryostat

To confirm effective knock out of Arc using AAV5-hSyn-mCherry-Cre, Arc cKO mice were injected at P60 and sacrificed at P74, then perfused with PBS followed by 4% ice-cold paraformaldehyde in PBS. The brain was removed and stored in 4% paraformaldehyde in PBS at 4C for 24h before being transferred to 30% sucrose in PBS at 4C until saturated. The brain was cut into 40 μm sections on a Leica 1950 CM cryostat and stored in PBS at 4°C. Sections containing binocular V1 were blocked in 5% normal donkey serum (Jackson ImmunoResearch) and 0.1% Triton X-100 (Amresco) in PBS for 1 h and then transferred into 1:1000 rabbit anti-Arc antibody (Proteintech) diluted in blocking buffer overnight. Sections were then washed 3 times for 10 min each time in PBS, before being placed in secondary donkey anti-rabbit Alexa Fluor 488 (Jackson ImmunoResearch) for 4 h. Sections were then washed 3 times for 10 min each time in PBS before mounting on slides with ProlongGold Antifade reagent (Invitrogen).