Immunofluorescence

The brain was fixed in 4% phosphate-buffered paraformaldehyde overnight at room temperature and then transferred to 4% phosphate-buffered paraformaldehyde containing 30% sucrose for cryopreservation at 4°C. Free-floating cryostat sections (20 μm) were obtained and preserved in 48-well plates containing 10 mM PBS at 4°C (WT-control: n = 5, 16p11.2del: n = 5; Δdisc1: n = 6). The sections were washed three times (5 min each) in 10 mM PBS (pH 7.4) on a slow orbital shaker. Subsequently, nonspecific blocking was performed in normal 5% normal goat serum (Vector Labs, Malvern, PA, US. #S-1000; RRID:AB_2336615), prepared in 10 mM PBS + 0.03% Triton-X100, for 1 hr at room temperature. The sections were incubated overnight at 4°C in primary antibody—rabbit anti-SK2 antibody (Thermofisher Scientific #PA5-41071)—diluted in blocking solution (10 mMPBS + 0.03% Triton-X 100 and 5% normal goat serum). Subsequently, the sections were washed two times in 10 mM PBS and incubated in a secondary antibody—goat anti-rabbit Alexa 488 (Thermofisher Scientific #A-11034; RRID:AB_2576217)—diluted in the blocking solution. Secondary antibody incubation was done for 1 hr at room temperature, with gentle shaking (35 rpm). Immunolabeled sections were washed and mounted on gelatin-coated slides using ProLong™ Diamond Antifade Mountant (Thermofisher Scientific #{"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}}P36970).