Rabbit anti hif 1α polyclonal antibody

The cultured NSCs were fixed in 4% paraformaldehyde solution (PFA) for 20 min, and 3 samples for each group were used for immunostaining. After three washes with PBS, the cell coverslips were permeabilized with 0.3% (v/v) Triton X-100 (T8787) for 30 min, rinsed, and blocked with 5% normal goat serum (Sigma-Aldrich) and 5% bovine serum albumin (BSA, Sigma-Aldrich) for 2 h. Then, the cells were incubated with the primary antibodies at 4℃ overnight. The following primary antibodies were used: mouse anti-nestin monoclonal antibody (1：200, Millipore), rabbit anti-SOX2 polyclonal antibody (1：200, Abcam, UK), rabbit anti-GFAP polyclonal antibody (1：200, Abcam), mouse anti-Tuj1 monoclonal antibody (1：200, Abcam), rabbit anti-HIF-1α polyclonal antibody (1：400, Abcam). All primary antibodies were diluted in 0.01 M PBS plus 2% BSA. For negative control, slides were incubated in PBS instead of primary antibodies. After washing, the cell coverslips were incubated with the following secondary antibodies: Alexa Fluor 488 donkey anti-mouse IgG (1：500; Invitrogen), Alexa Fluor 594 goat anti-rabbit IgG (1：500, Invitrogen). The nuclei were visualized by DAPI (H-1200, Vector, Burlingame, California, USA) staining. Immunostained positive cells were observed using a BX51 fluorescent microscope equipped with a DP70 digital camera (both from Olympus).