Oyster550

For immunocytochemical stainings (ICC) and for Western blots (WB) following primary antibodies were used from rabbit: anti-CDK5 (WB 1:1000, C-8 Santa Cruz), anti-homer1 (ICC 1:1000, Synaptic Systems), anti-VGLUT1 (ICC 1:1000, Synaptic Systems), anti-VGAT (ICC 1:1000, Synaptic Systems), anti-VGAT lumenal domain Oyster550-labeled (ICC 1:200, Synaptic Systems), from mouse: anti-synaptotagmin1 lumenal domain Oyster550-labeled (ICC 1:250, Synaptic Systems), anti-β-tubulin isotype III (WB 1:2000, Sigma), anti-Aβ_17–24 (4G8) (5 μg/ml, Signet), and from guinea pig: anti-synaptophysin (ICC 1:1000, Synaptic Systems). For ICC Alexa Fluor 488- (1:2000), Cy3- (1:2000) and Cy5- (1:1000) fluorescently labeled secondary antibodies were purchased from Jackson ImmunoResearch. For WB secondary antibodies labeled with Alexa Fluor 680 (1:20,000, ThermoFisher Scientific/Molecular Probes) and IRDye 800CW (1:20,000, Rockland) were used.

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.

Transverse sections (70 μm thick) through the MDH from the same 3 male Sprague–Dawley rats were immersed in 50% ethanol for 30 min to facilitate antibody penetration [46 (link)]. Immunofluorescent staining for the glycine transporter GlyT2, glutamic acid decarboxylase GAD (both isoforms), gephyrin, and PKCγ was carried out by incubating sections for 2 days at 4 °C in the following mixture of primary antibodies: goat anti-PKCγ (a gift from M Watanabe; 1:1000), rabbit anti-GlyT2 (a gift from F Zafra; 1:1000), mouse monoclonal anti-GAD65 (GAD6, Developmental Studies Hybridoma Bank, University of Iowa; 1:100), and mouse monoclonal anti-GAD67 (Merck, Watford, UK, catalog number mAb5406; 1:5000). These were revealed by overnight incubation in species-specific secondary antibodies raised in donkey and conjugated to Rhodamine Red, Alexa488, and Alexa647 (Jackson Immunoresearch), respectively. To avoid binding of the mouse secondary antibody to primary antibodies used in the subsequent step, a Fab’ fragment of donkey anti-mouse IgG (conjugated to Alexa 647) was used. The sections were then incubated overnight in mouse monoclonal antibody against gephyrin (clone 7a), conjugated to the fluorescent dye Oyster550 (catalog number 147 011C3, Synaptic Systems; 1:500), rinsed, and mounted in anti-fade medium and stored at −20 °C.