Rabbit anti collagen 3 antibody

Tissue sections (5 μm) were made as described above. After deparaffinizing, rehydrating, antigen retrieval, and blocking, the sections were incubated overnight at 4°C with rabbit anti-collagen I antibody (Abcam), rabbit anti-collagen III antibody (Abcam), rabbit anti-fibronectin antibody (Abcam), and rabbit anti-alpha smooth muscle actin (α-SMA) antibody (Abcam), respectively. On the next day, samples were washed with phosphate-buffered saline (PBS) 3 times for 5 min each time, before incubation with goat anti-rabbit secondary antibody labeled with horseradish peroxidase (KeyGEN Biotechnology) for 30 min. Diaminobenzidine (DAB) (KeyGEN Biotechnology) was used for coloring. Immunohistochemical assay of these outcomes was performed using Image Pro Plus software.

Total protein from skin tissue and BJ cells was extracted using RIPA buffer (Beyotime Institute of Biotechnology) and then quantified using a Pierce™ BCA Protein assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). A total of 40 µg protein/lane was separated via SDS-PAGE (Mini-Protean-3; Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene difluoride membranes (EMD Millipore). The membrane was blocked with 5% skimmed milk powder solution for 1 h and then incubated with the following primary antibodies at 4˚C overnight: Rabbit anti-collagen I antibody (cat. no. ab34710; 1:2,000 dilution; Abcam), rabbit anti-collagen III antibody (cat. no. ab7778; 1:5,000 dilution; Abcam), rabbit anti-MMP-1 antibody (cat. no. ab137332; 1:1,000 dilution; Abcam), rabbit anti-α-SMA antibody (cat. no. YM-H0645; 1:500 dilution; Shanghai Yuan Mu Biotechnology Co., Ltd.) and rabbit anti-β-actin antibody (cat. no. ab8227; 1:2,00 dilution; Abcam). The protein bands were then incubated with secondary antibody goat anti-rabbit IgG (cat. no. ab6721; 1:2,000 dilution; Abcam) at room temperature for 2 h and treated with enhanced chemiluminescence solution (Thermo Fisher Scientific, Inc.). β-actin was used as the internal reference and the gray values of protein bands were quantitatively analyzed by ImageJ software (version 1.46r; National Institutes of Health).