Ab75838

For the antibody shift assay (ASA), samples were pre-incubated for an hour with mouse anti-GABAB1 (Abcam, ab55051, Cambridge, UK; 1/5000) and rabbit anti-GABAB2 (Abcam, ab75838, Cambridge, UK; 1/5000), with gentle shaking at 4°C, followed by loading the mixture onto the BN gel. The lanes from the BN gel were cut out and the gel strips were equilibrated for 30 min in equilibration buffer [1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol] with gentle shaking and then briefly rinsed twice with SDS-PAGE electrophoresis buffer [25 mM Tris–HCl, 192 mM glycine and 0.1% (w/v) SDS; pH 8.3] and subsequently by Milli-Q water. SDS-PAGE was performed in a PROTEAN II xi Cell using 4% stacking gel and 10% separating gel. The gel strips were run at 12°C with an initial current of 50 V (during the 1st h). The voltage was subsequently increased to 100 V for the next 12 h (overnight) and then increased to 150 V until the dye front reached the bottom of the gel. The gel was then further subjected to a western blotting analysis as described earlier.

After voltage or current recording, the oocytes were washed twice with the modified Barth's solution and fixed in a 4% paraformaldehyde solution for 45 min at room temperature followed by washing with the Barth's solution. The oocytes were treated in 1% Triton‐X100 contained Barth's solution for 20 min, immersed in the blocking solution containing 5% goat serum for 2 h, then incubated overnight at 4°C in solution containing rabbit monoclonal anti‐GBRII (GABA_B receptor II, Abcam, ab75838, 1:1000) and 5% goat serum. After washing with the Triton‐X100 solution the oocytes were incubated with goat‐anti‐rabbit Cy‐3‐conjugated secondary antibody (1:600) for 1 h in darkness. Immunostained oocytes were visualized in a Zeiss LSM 700 confocal microscope system (Munich, Germany).

Chicks on day 0 were transcardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS) under deep anesthesia using a ketamine-xylazine cocktail. The brains were post-fixed with the same fixative for 24 h and immersed in 30% sucrose in PBS. The brain tissues were then cut into 18-μm-thick sections using a cryostat. For fluorescent staining, the sections including the IMM (Kuenzel and Masson, 1988 ) were blocked with 3% normal pig serum for 1 h and incubated with anti-GABA-A receptor subunit alpha 1 goat polyclonal antibody (sc-31403, 1:250; Santa Cruz Biotechnology, Santa Cruz, CA, United States) and anti-GABA-B receptor subunit 2 rabbit monoclonal antibody (ab75838, 1:250; Abcam plc) for 24 h at 4°C. The sections were then incubated with Alexa Fluor 546-conjugated anti-goat antibody (1:250; Thermo Fisher Scientific K.K., Waltham, MA, United States), Alexa Fluor 488-conjugated anti-rabbit antibody (1:250; Thermo Fisher Scientific K.K.), and Hoechst 33342 (Thermo Fisher Scientific K.K.). Fluorescent images were obtained using a confocal microscope (FV-10i; Olympus, Tokyo, Japan).

The IHC staining distribution pattern of GABABR (Sheilabi et al., 2018 (link)) proteins was performed as previously reported. Brain sections were dewaxed with xylene and rehydrated using a reduced concentration of alcohol. The sections were placed in a repair cassette filled with citrate antigen repair buffer (pH 6.0) for microwave antigen repair then washed for 15 min and incubated with 0.3% H₂O₂ in phosphate buffer saline (PBS) for 25 min. Subsequently, the sections were rinsed three times with PBS (pH 7.4) for 5 min each, and the tissue was covered uniformly with 3% bovine serum albumin (BSA) dropwise for 30 min. GABABR2 (ab75838, 1:50; Abcam, Cambridge, United Kingdom) and NR2B antibodies (21920-1-AP, 1:100; Proteintech, Rosemont, IL, United states) were diluted in TRIS buffered saline solution (TBS) containing 1% BSA overnight at 4°C. Afterward, sections were washed in PBS and incubated with secondary antibodies for 50 min. After being washed thrice in PBS, the slices were incubated for 5–15 min with 0.05% diaminobenzidine tetrahydrochloride (DAB) solution in 50-mM Tris buffer (TB) containing 0.01% H₂O₂ at 22–25 °C for 10–12 min with controlled color development time under a microscope (Peyvandi et al., 2021 (link)). The slices were then counterstained for 3 min with hematoxylin, dehydrated with ethanol and xylene, and sealed.