Goat anti mouse alexa fluor 647

The cultured astrocytes and neurospheres were washed and fixed with 4% paraformaldehyde at 4°C for 30 min. The astrocytes were incubated overnight at 4°C with the following primary antibodies: mouse anti-GFAP (1:300; Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-HMGB1 antibodies (1:100; Abcam, Cambridge, MA, USA). Neurospheres were incubated overnight at 4°C with the following primary antibodies: rabbit anti-nestin (1:50; Proteintech, Wuhan, China) and mouse anti-Sox-2 antibodies (1:100; Cell Signaling Technology). The cells were then incubated with secondary antibodies, including goat anti-mouse Alexa Fluor 647 (1:200; Beyotime Institute of Biotechnology), goat anti-rabbit TRITC (1:100) and goat anti-mouse FITC (1:100) (all from Beijing Ding Guo Changsheng Biotech Co., Ltd., Beijing, China) for 1 h at 37°C in the dark. Finally, the cells were examined by laser-scanning confocal microscopy on an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan) equipped with a FluoView FVX confocal scan head (Leica Microsystems GmbH, Wetzlar, Germany).

Cells seeded on glass slides were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% TritonX-100 for 10 min and then blocked with 5% BSA for 2 h at room temperature. Next, the cell slides were incubated with primary antibodies at 4 °C overnight. Then, the cell slides were washed with PBST three times and incubated with fluorescent-conjugated secondary antibodies-Goat anti-Rabbit Alexa Fluor 488 or Goat anti-Mouse Alexa Fluor 647 (Beyotime Institute of Biotechnology, Jiangsu, China) for 2 h at room temperature away from the light. Finally, the nucleus were stained with DAPI for 5 min. Fluorescence was visualized under laser confocal microscopy.