A21206

Primary mouse cortical neurons (ThermoFisher A15586) were purchased and plated onto poly-L-lysine-coated coverslips in 12-well plates containing plating medium (MEM with L-glutamine, 10% horse and fetal bovine serum, pen strep, and 25 mM glucose). Plating medium was replaced with Neurobasal medium, B-27 supplement, and GlutaMAX supplement after 24 h, and cells were fed every 3 d. At 12 days in vitro, mouse cortical neurons were washed twice with Dulbecco's phosphate-buffered saline (D-PBS), fixed in 4% paraformaldehyde for 15 min at room temperature (RT), and rinsed three times with D-PBS. Neurons were permeabilized with 0.3% Triton ×100 and blocked in 5% goat serum for 1 h at RT. Cells were washed three times in D-PBS and incubated overnight at 4 °C with the primary antibody. The following primaries were used: anti-TSG101G (rabbit, Abcam ab125011, 1:50), anti-TOMM20 (mouse, Abcam ab56783, 1:100), and anti-EEA1 (mouse, Santa Cruz sc-137130, 1:100). Cells were washed and incubated with donkey Alexa-488–conjugated anti-rabbit (Abcam a21206, 1:1,000) and goat Alexa-568–conjugated anti-mouse (Abcam, ab175473, 1:1,000) secondary antibodies for 1 h at RT. Cells were washed three times with D-PBS, counterstained with Hoechst for 30 min at RT, rinsed, and mounted with Prolong Gold antifade solution.