Anti gfap

The cells that migrated out after 48 h of DRG culture were taken, and 4% paraformaldehyde (Beyotime, Shanghai, China) was added to fix the cells. After the fixation, the cells were washed 3 times with PBS and permeabilized with 0.5% Triton^® X-100 (Beyotime, Shanghai, China) at room temperature for 20 min. After aspirating the cleaning solution, 5% goat serum (Beyotime, Shanghai, China) was added and blocked at room temperature for 30 min. Then, anti-GFAP (Beyotime, Shanghai, China) as the primary antibody at a dilution of 1:10,000 was incubated overnight at 4°C and Cy3-labeled secondary antibody (Beyotime, Shanghai, China) at a dilution of 1:20,000 was incubated at room temperature without light for 1 h. After excessively washing with PBS, the cell nuclei were stained by DAPI and observed under a fluorescence microscope (Olympus, Tokyo, Japan).

Immunostaining was performed following the previously described method. Briefly, cryosections were permeabilized with 0.5% Triton X-100 in PBS for 15 min, blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, and then stained with primary antibodies in blocking solution overnight at 4 °C prior to incubation with secondary antibodies diluted in blocking solution for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The primary antibodies were as follows: mouse anti-β-III-tubulin (1:100, Beyotime), rabbit anti-glial fibrillary acid protein (anti-GFAP; 1:100, Beyotime), rabbit anti-TNF-α (1:100, Beyotime), and IL-1β (1:100, Beyotime). To quantify immunofluorescence intensity, the areas of β-III-tubulin and GFAP immunopositivity were determined by thresholding based on the images obtained using Image J (National Institutes of Health, Bethesda, MD, USA). TNF-α- and IL-1β-positive cells were quantified from at least three sections of each rat. Each group included six rats. Comparisons between groups were made using one-way analysis of variance (ANOVA).