Gb11226

Paraffin sections were dewaxed and placed in a buffer filled with citric acid antigen repair buffer (pH = 6.0) for antigen repair. Sections were slightly dried and incubated with a histochemical pen by drawing circles around the tissue and adding drops of BSA blocking solution inside the circles for 30 min at room temperature. Sections were incubated with primary antibodies (anti-NF200: bs-10680r, anti-MBP: GB11226, Servicebio, China) respectively and kept flat overnight in a wet chamber at 4℃. Sections were washed with PBS for 3 × 5 min and then incubated with secondary antibodies for 50 min at room temperature in the dark. Subsequently, sections were washed with autofluorescent bursting agent for 5 min and rinsed with tap water for 10 min before staining with DAPI stain (G1012, Servicebio, China) to label the nuclei. Slices were sealed with anti-fluorescence quenching sealer (G1401, Servicebio, China). Images were observed and acquired under a Confocal laser scanning microscope (CLSM Zeiss). The intensity of expression of NF200 or MBP positive areas in the injury centre was assessed using ImageJ to quantify the expression of NF200 or MBP.

The Schwann cells were co-cultured with BDNF for 3 days and then the cells were harvested for western blot analysis. And then the Schwann cells protein extracts were prepared by Total Protein Extraction Kit (BestBio, China). All the cell proteins were mixed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer (P0015, Beyotime) and heated at 100°C for 5 min. Protein samples (80 μg/well) were loaded on SDS–PAGE (10–12%) and electroplated onto polyvinylidene fluoride (PVDF) films (Millipore, United States). The PVDF films were blocked with 5% non-fat milk (BD, United States) for 60 min at room temperature and subsequently incubated overnight at 4°C with the primary antibodies: anti-PCNA antibody (GB11010, Servicebio), anti-MBP antibody (GB11226, Servicebio), and rabbit β-actin Rabbit antibody (GB11001, Servicebio). After incubating the PVDF films with horseradish peroxidase (HRP)-labeled secondary antibodies (GB23303, Servicebio) for 60 min at 25°C, the signal was collected by Image Studio Digits Ver 4.0. Density values were normalized to β-actin. The quantification of Western blot data was performed using Image-J software.