Live-cell surface labelling and immunocytochemistry

Live cell surface labeling was performed for cells expressing 3xF_M-GluA1 or 4xF_M-Nlgn1 using anti-HA (Biolegends, clone 16B12 #901501 RRID:AB_2565006; 1:1000) or for experiments using 4xF_M-VSV-G using anti-VSV-G (Kerafast, clone 8G5F11, 1:1000). Cells were incubated in NBA + B27 with antibody at 37°C for 15 min. Cells were washed once with artificial cerebrospinal fluid (ACSF) solution containing the following (in mM): 130 NaCl, 5 KCl, 10 HEPES, 30 glucose, 2 CaCl₂, 1 MgCl₂,. 002 TTX, pH 7.4. Cells were then fixed with 4% PFA for 10 min at room-temperature and labelled with fluorescent-conjugated secondary for 30 min in non-permeabilizing conditions. For immunocytochemistry, cells were fixed in 4% PFA, permeabilized with 0.1% Triton X-100 or 0.5% Tween-20 for 10 min and blocked for 30 min at room temperature with 5% BSA. Cells were incubated with primary antibodies at the reported dilutions for 60 min at room temperature, washed in PBS, incubated with fluorescent-conjugated secondary antibodies for 60 min at room temperature and mounted (Prolong Gold, Life technologies). Primary antibodies used in this study include BIP (abcam; ab21685 RRID AB_2119834; 1:1000), GM130 (BD; 610822 RRID AB_398141; 1:1000), TGN38 (clone 2F7 was a gift from Kathryn Howell), mCh (abcam; 167453 RRID AB_2571870; 1:1000), GFP (Invitrogen; A11122 RRID AB_221569; 1:1000 or neuromab; clone N86/6 RRID AB_2313651; 1:1000) and ERGIC/p58 (Sigma; E1031 RRID AB_532237, 1:500). Fluorescent-conjugated secondary antibodies include goat anti-mouse alexa fluor 647 and goat anti-rabbit alexa fluor 647 or alexa fluor 568 (Life technologies, 1:1000)