Ab7260

Total protein content was extracted from the cultured cells or tissues, and protein concentrations were determined using the Pierce BCA assay kit (Beyotime, Haimen, China; P0012S). Antibodies against GFAP (1:10,000; Abcam, Cambridge, UK; ab7260), NDRG2 (1:5000; Sigma-Aldrich; HPA002896), p-STAT3 (1:2000; CST, Naples, FL, USA; 9145P), STAT3 (1:2000; CST, 4904 T), p-Akt (1:2000; CST, 4060S), Akt (1:2000; CST, 9272S), c-Jun (phospho S63) (1:1000, CST, 91952 T), c-Jun (1:1000, CST, 9165 T), TNFα (1:500, Abcam, ab6671), HIF1α (1:1000, Sigma, SAB5200017), GAPDH (1:2000; CMC-TAG, AT0002) and β-actin (1:2000; CMC-TAG, AT0001) were used for western blot analysis. The band intensities of the target proteins were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA). GAPDH and β-Actin were used as the loading control. The results were derived from three independent experiments.

Mice were sacrificed by cervical dislocation and the brains immediately removed, post-fixed overnight in 4% paraformaldehyde (PFA) and then cryoprotected in phosphate buffer saline (PBS) containing 30% sucrose for 2 days. The brains were then cut into 40 μm free floating coronal sections (one in 12 series) using a vibratome and transferred to a 24-well plate for immunostaining. Following 3 washes with 0.2% Triton X-100 in PBS (PBST), the sections were incubated in blocking solution containing 5% goat serum in PBST for one hour and then incubated with the primary antibodies (GFAP Abcam ab7260 1:1000, S100B Sigma-Aldrich S2352 1:500, D2R Santa Cruz SC5303 1:200, D2R Merck Millipore AB5084P), in blocking serum overnight at 4 °C. After three five-minute washes in PBST, the sections were incubated for 2 h with the appropriate secondary antibodies at room temperature in the dark. Following three more washes, sections were incubated for 10 min with DAPI (1:1000, Sigma-Aldrich). Finally, sections were washed a further 3 times in PBST and then mounted using vectashield onto 1% gelatin- coated slides for imaging. As a negative control, some sections were incubated without the primary antibody.

Cultured cells were fixed 4% paraformaldehyde, permeabilized with 0.15% Triton X-100 in PBS (PBST). blocked with 3% FBS in PBST, followed by incubation with the following primary antibodies overnight at 4 °C: anti-GFP (Abcam, Cambs, UK; ab13970; 1:1000), anti-Tuj1 (Abcam, Cambs, UK; ab78078; 1:1000), anti-TH (Abcam, Cambs, UK; ab112; 1:1000 or Santa Cruz, Heidelberg, Germany; sc-25269; 1:100), anti-GFAP (Abcam, Cambs, UK; ab7260; 1:1000), anti-S-100β (Sigma, Saint Louis, MO, USA; S2532; 1:250), anti-Nestin (Abcam, Cambs, UK; ab134017; 1:1000), anti-PAX6 (Abcam, Cambs, UK; ab195045; 1:500), anti-MAP2 (Sigma, Saint Louis, MO, USA; M4403; 1:1000 or Abcam, Cambs, UK; ab32454, 1:200). Immunoreactivity was visualized using appropriate Alexa Fluor-conjugated secondary antibodies and observed using the confocal microscope (Nikon, Tokyo, Japan, A1R HD25). The cell nuclei were visualized by staining with DAPI (Beyotime, Shanghai, China; C1002) at 0.5 μg/mL for 10 min.