Anti mouse antibody

The proteins of primarily cultured NSCs or spinal cord tissues from SCI rats were isolated by RIPA lysis buffer (Beyotime, Shanghai, China), and the concentrations were determined by a BCA detection kit (Beyotime). About 20ug proteins were loaded and electrophoresed onto 12% SDS polyacrylamide gel (GE Healthcare Bioscience, Marlborough, MA, United States) and then transferred to polyvinylidene fluoride (PVDF) membrane (MilliporeSigma, Burlington, MA, United States). The membranes were blocked by 5% non-fat milk for 60 mins at 37°C and were then incubated with anti-NGF monoclonal antibody (1:1000, Abcam, Cambridge, MA, United States), anti-CREB (1:1000, Abcam), anti-GDNF (1:500, Abclonal, Wuhan, China), anti-BDNF (1:1000, Abcam), anti-VEGF (1:1000, Boster, Pleasanton, CA, United States) and anti-β-actin (1:5000, Boster) overnight at 4°C. The primary antibody incubation was followed by incubation with a secondary peroxidase-conjugated anti-rabbit (1:5000, Boster) or anti-mouse antibody (1:5000, Boster) at room temperature for 2 h. The detection of signal was conducted using Bryo ECL kit (Bytotime), and the measurement of proteins’ expression was done by Image J (Joonas “Regalis” Rikkonen, Version 1.8.0).

After continuous diet treatment for 28 days, the pancreas of the rat was taken and the surface blood was washed with physiological saline, fixed in 10% PBS-paraformaldehyde at 4°C for 24 hours. After paraffin embedding and sectioning (5 μm), tissues were stained with hematoxylin-eosin (H-E) and insulin expression in pancreatic tissue was observed with immunohistochemistry. In detail, paraffin-embedded sections were incubated with the primary anti-insulin antibody (1 : 200; host species : mouse; Boster, China) for 24 h at 4°C and subsequently decorated with the secondary antibody (anti-mouse antibody (1 : 200); Boster, China) for 1 h at room temperature (RT); then, reactions were developed using DAB (3,3′-diaminobenzidine) chromogen and DAB substrate buffer (Dako). All images were acquired with a Zeiss Axio Observer epifluorescence microscope.

Western blot was performed as we previously reported [25 (link)]. The antibodies used in this article are listed as follows: anti-p57 (1 : 500, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-p53 (1 : 500, Wanleibio, Xi'an, China), anti-p-p53 (1 : 500, Wanleibio, Shenyang, China), anti-GAPDH (1 : 5000; Genesci, Beijing, China), anti-PCNA (1 : 1000, Boster, Wuhan, China), anti-Cyclin A (1 : 300, Santa Cruz, Dallas, Texas, USA), anti-Cyclin E (1 : 300, Santa Cruz), anti-OCT4 (1 : 500, Santa Cruz), anti-NANOG (1 : 500, PeproTech, Rocky Hill, New Jersey, USA), anti-SOX2 (1 : 1000, Proteintech Group, Rosemont, Illinois, USA), horse-radish peroxidase-conjugated anti-rabbit antibody (1 : 3000, Boster), and anti-mouse antibody (1 : 2000; Boster).