Tcs sp8 2

Mouse primary cortical neurons in 15 mm culture dish after seven to nine days were fixed with 4% PFA for 15 min at room temperature after washed three times by 0.01 mol/L PBS before blocked by 10% sheep serum with 0.3% Triton X-100 (Amresco, Solon, OH, USA) in 0.01 mol/L PBS for 1 h. Cells were incubated overnight at 4 °C with primary antibodies including rabbit monoclonal NeuN antibody (1:1000, Abcam, ab177487), mouse monoclonal anti-KCa3.1 antibody (1:200, Alomone, ALM-051) and rabbit anti-Nav1.1 antibody (1:200, Alomone, ASC-001). To test neuronal purity of the primary cultured cortical neurons, we also used rabbit anti-GFAP (glial fibrillary acidic protein) antibody (1:200, Abcam, ab16997) for astrocytes; recombinant anti-Iba1 (ionized calcium-binding adaptor molecule-1) antibody [EPR16589] (1:500, Abcam, ab178847) for microglial cells; and rat mAb to anti-MBP (Myelin basic protein) antibody (1:100, Abcam, ab7349) for oligodendrocytes. The immunoreactivity was visualized with Alexa Fluor 488- or 594-conjugated secondary antibodies (1:500; ZSGB-BIO) and goat anti-rat IgG H&L (Alexa Fluor® 488) (1:500, Abcam, ab150165). The nuclei were stained by Hochest33342. Immunocytochemical staining was scanned by confocal microscopy (TCS SP8 II, Leica Microsystems, Wetzlar, Germany).