Rabbit anti gfap

When cells reached 70-80% confluence, they were fixed with 4% paraformaldehyde overnight at 4°C. The cells were then blocked and permeabilized for 1 h at 37°C in PBS with 2% bovine serum albumin (BSA; Thermo Fisher, USA) and 0.1% Triton X-100, followed by incubation with rabbit anti-nestin (Boster, China), rabbit anti-pax6 (Boster, China), rabbit anti-GFAP (Boster, China), rabbit anti-S100 (Boster, China), and rabbit anti-β-III tubulin (Applied Biological Materials Inc., Canada) at the appropriate dilution overnight at 4°C. After washing with PBS, the cells were incubated at 37°C for 2 h with appropriate secondary antibodies: CyTM3-conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson, USA). Nuclei were counterstained with DAPI. Images were captured using an Olympus inverted fluorescence microscope (IX73, Olympus, USA).

For immunohistochemistry and Fluoro-Jade C staining, three brain slices with the injured area of each mouse were randomly picked for each staining. The primary antibodies were used as follows: rabbit-anti-Arg1 (1:300, Boster, Wuhan, China, Cat# BA0056), mouse-anti-iNOS (1:300, BD Biosciences, Franklin Lakes, USA, Cat# 610328), and rabbit-anti-GFAP (marker for astrocytes, 1:300, Boster, Cat# BA3796-2). Followed by appropriate secondary antibodies, the sices were imaged under an epifluorescence (Olympus, BX51) or a confocal microscope (Olympus, Japan, FV1000). Three rectangles (200 μm × 100 μm) were randomly selected on the borders of the immunoreactive region of each slice, inwards from the interface between staining-positive and –negative tissue. Then the cell densities were analyzed by ImageJ software. To assess neurodegeneration, Fluoro-Jade C staining was performed as described previously. In brief, randomly selected slices were placed in an ethanol gradient and then incubated in a 0.06% potassium permanganate solution. Slices were immersed in 0.0001% Fluoro-Jade C solution (Merck, Billerica, USA, Cat# AG325) to stain the degenerative neurons. The slices were dried and cleared in xylene and then mounted in DPX. Three rectangles (200 μm × 100 μm) were randomly selected on the borders of the infarct region, where the density of FJC-positive cells was measured with Image J.