Rabbit anti gr antibody

For immunofluorescent assay, the cultured astrocytes were fixed with 4% paraformaldehyde in 10 mM phosphate-buffered saline (PBS; pH 7.4) for 20 min at room temperature, blocked with 3% normal goat serum (Sigma-Aldrich) for 30 min at room temperature, and incubated with rabbit anti-GR antibody (1:100, monoclonal, Cell Signaling, Boston, MA, USA) and mouse anti-GFAP antibody (1:200, monoclonal, Abcam, Cambridge, UK) overnight at 4°C. Then, the cells were washed with PBS, incubated with TRITC-conjugated goat anti-rabbit IgG (1:200; Vector, Burlingame, CA, USA) and FITC-conjugated goat anti-mouse IgG (1:200; Vector) secondary antibodies for 60 min while shaking at room temperature in darkness, and washed with PBS. Finally, the astrocytes were incubated for 2 min with nucleic acid stain, Hoechst33258 (Invitrogen, Carlsbad, CA, USA), diluted 1:5000 in PBS. All primary and secondary antibodies were diluted in PBS containing 1% normal goat serum. The images were captured using a confocal laser scanning microscopy (LSM 710, Carl Zeiss, Germany).