Streptavidin coated quantum dots qds

Neurons were incubated for 3–5 min at 37°C with primary antibodies against extracellular epitopes of GABA_AR α2 subunit (guinea pig, 1:750/1:1000, provided by J.M. Fritschy), washed, and incubated for 3–5 min at 37°C with biotinylated Fab secondary antibodies (goat anti–guinea pig, 4–12 μg/ml; Jackson Immunoresearch) in imaging medium. After washes, cells were incubated for 1 min with streptavidin-coated quantum dots (QDs) emitting at 605 nm (1 nM; Invitrogen) in borate buffer (50 mM) supplemented with sucrose (200 mM) or in PBS (1 M; Invitrogen) supplemented with 10% casein (vol/vol; Sigma-Aldrich). Washing and incubation steps were all done in imaging medium. To assess the membrane dynamics of GABA_AR α2 subunit at inhibitory synapses in neurons expressing the eGFP-DN mutant, inhibitory synapses were stained by incubating live neurons for 48 h at 37°C in a 5% CO₂ humidified incubator with a primary VGAT antibody directly coupled to Oyster550 (1:200, Synaptic Systems) diluted in conditioned maintenance medium.

Neurons were stained as described previously^{53 (link)}. Briefly, cells were incubated for 3–5 min at 37 °C with primary antibodies against Flag (mouse, 1:700, Sigma, cat #F3165), washed, and incubated for 3–5 min at 37 °C with biotinylated Fab secondary antibodies (goat anti-mouse: 1:700; Jackson Immuno research, cat #115-067-003, West Grove, USA) in imaging medium. After washes, cells were incubated for 1 min with streptavidin-coated quantum dots (QDs) emitting at 605 nm (1 nM; Invitrogen) in borate buffer (50 mM) supplemented with sucrose (200 mM) or in PBS (1 M; Invitrogen) supplemented with 10% Casein (v/v) (Sigma).