Immunocytochemistry (ICC)

Dissociated mouse hippocampal cultures were fixed with 4% paraformaldehyde in PBS, at 37°C, for 15 min. Fixed neurons were washed twice with PBS and then permeabilized and quenched, at RT, for 10 min in PBS with 0.2% saponin and 50 mM ammonium chloride. Samples were then incubated for one hour in blocking buffer 1 (PBS with 10% goat serum, 10% bovine serum albumin (BSA), 0.02% sodium azide (NaN₃), and 0.02% saponin), followed by a 1 hr incubation with primary antibodies diluted in blocking buffer 2 (PBS with 1% BSA, 0.02% NaN₃, and 0.02% saponin). Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID:AB_2085748), anti-MAP2 (1:100) (Millipore; AB15452, RRID:AB_805385), anti-MAP2 (1:500) (Millipore; AB5543, RRID:AB_571049), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID:AB_2092361), anti-vGlut1 (1:500) (Millipore; AB5905, RRID:AB_2301751), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID:AB_2199314), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID:AB_11042457), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID:AB_1210382). Samples were then washed four times in wash buffer (PBS with 0.02% NaN₃, and 0.02% saponin) and incubated for one hour in secondary antibody diluted in blocking buffer 2. Secondary antibodies used include, goat anti-chicken IgY-Alexa Fluor 405 (1:500) (abcam; ab175675, RRID:AB_2810980), goat anti-mouse IgG1 IgG-Alexa Fluor 488 (1:500) (Thermofisher; A-21121, RRID:AB_2535764), goat anti-guinea pig IgG-Alexa Fluor 488 (1:500) (Thermofisher; A-11073; RRID:AB_2534117), goat anti-rabbit IgG-Alexa Fluor 546 (1:500) (Thermofisher; A-11010, RRID:AB_2534077), goat anti-chicken IgG-Alexa Fluor 546 (1:500) (Thermofisher; A-11040, RRID:AB_2534097), goat anti-guinea pig IgG-Alexa Fluor 546 (1:500) (Thermofisher; A-11074, RRID:AB_2534118), goat anti-mouse IgG1 IgG-Alexa Fluor 647 (1:500) (Thermofisher; A-21240, RRID:AB_2535809), and goat anti-mouse IgG2b-Alexa Fluor 647 (1:500) (Thermofisher; A-21242; RRID:AB_2535811). After secondary antibody incubation, neurons were washed twice in wash buffer and three times in PBS before being mounted on glass slides with ProLong Diamond Antifade Mountant (Thermfisher; {"type":"entrez-protein","attrs":{"text":"P36965","term_id":"544168","term_text":"P36965"}}P36965). Images for Figure 1 were acquired on a Zeiss LSM 880 with a 63 × 1.4 NA oil immersion objective in confocal mode. Images for Figure 4 were acquired on the LSM 880 but using the Airyscan super-resolution detector; identical laser and gain settings were used for all samples. Images were deconvolved using automatic Airyscan settings. The same linear brightness and contrast adjustments were applied to all ICC images for publication.