Anti pcna antibody

Newly formed endothelial surface marker CD31 and proliferating cell nuclear antigen (PCNA) were detected by IF to assess angiogenesis and proliferation of the grafts. Briefly, the sections were blocked in a solution containing 0.2% TritonX-100 and 3% goat serum for 1.5 h and then incubated with anti-CD31 antibody (1:800, Servicebio, China) and anti-PCNA antibody (1:800, Servicebio, China) diluted in antibody diluent overnight at 4 °C followed by incubation with corresponding fluorescently labeled secondary antibody (1:400, Servicebio, China) at 37 °C for 1.5 h. DAPI was routinely used for nuclei counterstaining. The number of CD31-labeled neovascularization from each sample was calculated based on the five randomly selected regions of three discontinuous sections. Furthermore, we applied double-staining with CM-Dil and CD31 to analyze the differentiation of ADSCs in 3D-bioprinted engineering ovary group, and the fresh-frozen sections were obtained from the samples at 4 weeks after transplantation. The specific staining method was performed as described previously. Fluorescent images were acquired using an optical microscope (Zeiss, Germany).

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.