Visualizing Surface GABA(A) Receptors

The labeling of surface GABA_A receptors and confocal immunofluorescence microscopy analysis were performed according to published procedure [14 (link)]. Briefly, cells on coverslips were fixed for 3 min with 4% formaldehyde on ice and incubated in 100 μL of HEPES buffer (HEPES 25 mM, NaCl 140 mM, KCl 5.4 mM, CaCl₂ 1.8 mM, glucose 15 mM, pH = 7.4) containing mouse monoclonal anti-α1 antibody (Millipore #MAB339) and rabbit monoclonal anti-Na⁺/K⁺-ATPase, a plasma membrane marker (Abcam #ab76020), or rabbit polyclonal anti-γ2 antibody (Synaptic Systems #224,003) and mouse monoclonal anti-Na⁺/K⁺-ATPase, a plasma membrane marker (Santa Cruz Biotechnology #sc-48345) for 1.5 h. The primary antibodies were used at 1:300 dilutions. Cells were then incubated with 100 μL of HEPES buffer containing an Alexa 488-conjugated secondary antibody (1:600) and an Alexa 594-conjugated secondary antibody (1:600). Afterwards, cells were permeabilized with saponin (0.2%) for 10 min and incubated with DAPI (1 µg/mL) for 3 min to stain the nucleus. The coverslips were then mounted and sealed. For confocal immunofluorescence microscopy, an Olympus IX-81 Fluoview FV1000 confocal laser scanning system was used. A 60X objective collected images using FV10-ASW software. Quantification of the fluorescence intensity after background correction was done using the ImageJ software from the NIH.