Anti nkcc1

ATX (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 98% pure) was dissolved in dimethyl sulfoxide (DMSO); the DMSO content in every group was 0.1%. Pyrrolidine dithiocarbamate (PDTC), a NF-кB inhibitor, was purchased from Beyotime (Jiangsu, China) and used at a concentration of 10 μmol/L. Bumetanide, a NKCC1-specific inhibitor, was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used at a concentration of 50 μmol/L [22 (link)]. Anti-NKCC1, anti-glial fibrillary acidic protein (GFAP) and anti-NF-кB/p65 antibodies were purchased from Millipore (Billerica, MA, USA) and Santa Cruz Biotechnology, respectively. Anti-pro caspase 3 antibody was purchased from Abcam (Cambridge, MA, USA). Antibodies against Bcl-2, Bax, cleaved caspase-3, IL-1β, IL-6, TNF-α, β-actin, β-tubulin, and Histone H3 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Brain slices were post-fixed with 10% formalin, washed three times with PBS, incubated for 30 min with 0.3% Triton, and then blocked with 1% goat serum for 1 h to avoid binding of non-specific antibodies. Sections were then incubated at 4°C overnight with the following primary antibodies: anti-NKCC1 (Millipore, PA5-118800, Boston, MA, USA, 1:200), anti-NeuN (Sigma-Aldrich, St. Louis, MO, USA, MAB377, 1:200). Secondary antibodies conjugated to Alexa Fluor 488 or 594 (1:1,000; Invitrogen) were applied for 1.5 h at room temperature. Slides were mounted with Fluoroshield containing DAPI (Ab104139; Abcam) and observed with a confocal microscope (LSM710; Zeiss, Oberkochen, Germany).