Mouse anti microtubule associated protein map2

Neuronal cultures were fixed for 15 min (4% paraformaldehyde), permeabilized for 15 min (0.1% Triton-X 100, 0.1% BSA) and blocked for 1 h at room temperature (0.1% BSA, 1% goat serum in PBS). Coverslips were then immunostained with rabbit anti-KCC2 (1:1000; Protein Tech, Rosemont, IL) and mouse anti-microtubule-associated protein (MAP2) (1:500; Millipore, Burlington, MA) for KCC2 immunoreactivity and differentiation studies. Rabbit anti-MAP2 (1:500; Abcam, Cambridge, UK), mouse anti-glial fibrillary acidic protein (GFAP) (1:1000; Millipore), rabbit anti-Na⁺-K⁺-Cl⁻ cotransporter 1 (NKCC1) (1:100; Abcam), mouse anti-MAP2, mouse anti-glutamate decarboxylase (GAD) 67 (1:500; Abcam), rabbit anti-glutaminase (1:500; Abcam), and mouse anti-MOR (1:500; Antibodies Incorporated, Davis, CA). were used for differentiation studies. Alexa Fluor 488 and 594-conjugated secondary antibodies (1:1000; Thermo Fisher, Waltham, MA) were used for all studies as well as Hoechst 33342 (1:10000; Invitrogen, Carlsbad, CA) to detect nuclei. Cells were visualized and images obtained using a Zeiss LSM 700 confocal module configured to an Axio Observer Z.1 and Zen 2010 software (Zeiss Inc., Thornwood, NY). Cells were manually counted using the CellCounter plugin for Image J. At least 200 Hoechst+ cells were counted per sample.