Ab193311

Brains (n = 6/treatment group from different litters generated in Experiment 2) were processed for immunohistochemistry. Coronal cryostat sections (12 μm) including the hippocampus (CA1, CA2, CA3) and the basolateral amygdala were mounted as contiguous triplicates. Sections were fixed in cold methanol (−20 °C), blocked with 5% goat serum, 0.3% Triton X-100 in phosphate buffered saline (PBS) for 2h at 4 °C, followed by an overnight incubation at 4 °C in primary antibody against either parvalbumin (1:500, ab11427; abcam), MAP2 (1:500, ab32454; abcam), GABA Aα1 (1:500, ab33299; abcam), GABA Aα2 (1:500, ab193311; abcam) or GABA B1 (1:500, ab55051; abcam) in PBS with 1% bovine serum albumin (BSA), 0.3% Triton X-100. Sections were incubated with Alexa Fluor 555 anti-rabbit IgG, Alexa Fluor 488 anti-mouse IgG or Alexa Fluor 568 anti-mouse IgG secondary antibodies (Thermo Fisher Scientific) at 1:500 for 2h at 4 °C. Vectashield Mounting Medium with DAPI (Vector Laboratories, USA) was used to mount coverslips.

The rats were sacrificed 24 h after the 2nd injection after MWT assessment was completed. The rats were anesthetized with 2% pentobarbital sodium (40 mg/kg, i.p.). Then, the rats were quickly perfused with 0.9% NaCl (4°C) followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for prefixation. The ipsilateral L4–L6 DRGs were removed and postfixed in 4% paraformaldehyde for 3 h at 4°C before being transferred to 15 and 30% sucrose for dehydration and stored in a −80°C freezer. The DRGs were transversely sectioned (10 μm) with a cryostat and dried at 37°C for 20 min. Then, the sections were blocked with 5% normal donkey serum in TBST (1% Tween-20) for 1 h at 37°C. Then, the sections were incubated with primary antibodies diluted in blocking solution overnight at 4°C. The primary antibodies were mouse anti-GABAARα2 (ab193311, Abcam, Cambridge, United Kingdom) and rabbit anti-PKCε (ab124806, Abcam, Cambridge, United Kingdom). Then, the sections were washed with TBST and incubated with donkey anti-rabbit (Alexa Fluor 488-labeled) or goat anti-mouse (Cy3-labeled) secondary antibody at 37°C for 1 h (diluted with 5% normal donkey serum in TBST). Images were taken using an Imager. M2 microscope (ZEISS, Germany). The analysts were blind to the experiment design.