Aga 014

Dorsal hippocampi were collected around the tips of the hippocampal cannulas. Tissue was lysed in modified RIPA buffer, incubated 15 min on ice, and centrifuged for 15 min (15,000g) at 4 °C. Samples were subjected to SDS-PAGE (10 μg per well) and transferred to PVDF membrane (Biorad). Membranes were blocked with I-block (Tropix), incubated with primary antibody overnight at 4 °C, and with secondary antibody for 1 h at room temperature. Primary antibodies used were against GABA_AR-α1 (1:500, Sigma-Aldrich, G4416), GABA_AR-α4 (1:500, sc-20917, Santa Cruz), and GABA_AR-δ (1:500, Alomone, AGA-014). Secondary antibodies were DyLight 800 (Bio Rad). Bands were visualized using ChemiDoc MP Imaging System (Bio-Rad). All antibodies gave bands at the predicted molecular sizes. Specific band intensities were normalized to the total protein bands intensity of each lane.

Mice were anesthetized with an i.p. injection of 240 mg/kg Avertin and transcardially perfused with ice-cold 4% paraformaldehyde in phosphate buffer (pH 7.4, 150 ml per mouse). Brains were removed and post-fixed for 24 h in the same fixative and then immersed for 24 h each in 20 and 30% sucrose in phosphate buffer. Brains were frozen and 50 μm sections were cut for use in free-floating immunohistochemistry (Jovasevic et al., 2015 (link)) with primary antibodies against mCherry (1:1000, rabbit, Abcam, AB167453), Tyrosine hydroxylase (1:2000, Immunostar, 22941), Calretinin (1:4,000, Swant, CG1), GABA_AR-δ (1:2000, Alomone, AGA-014). Secondary antibodies were obtained from Jackson ImmunoResearch (1:200, Alexa Fluor® 594 AffiniPure Donkey Anti-Mouse IgG [H+L]). GFP was visualized by its intrinsic fluorescence. Nuclei were counterstained with Hoechst 33342 (ThermoFisher), except in double staining experiments, where blue color was used for one of the antibodies. Sections were mounted using FluorSave (Millipore-Sigma) and observed with Leica microscope equipped with a CCD (Olympus) camera, using 5 ×, 10 ×, or 20 × objectives. For light microscopy, signals were visualized with diaminobenzidine (Sigma).