Rabbit anti collagen type 3

PAFs were seeded on coverslips in 6‐well plates to 60% to 70% confluence before they were made quiescent in serum‐free medium for 24 hours and then incubated in DMEM containing 0.5% FBS. The cells were stimulated with or without TGF‐β1 (10 ng/mL) for 72 hours. After treatment, the cells were fixed in 4% formaldehyde for 15 minutes at room temperature and blocked and permeabilized in 5% bovine serum albumin with 0.3% Triton X‐100 (Sigma‐Aldrich) for 30 minutes at 37°C. Coverslips were incubated with rabbit anti–collagen type I (Abcam) or rabbit anti–collagen type III (Sigma‐Aldrich) antibody at 4°C overnight. The coverslips were then washed 3 times with PBS and incubated with FITC‐conjugated goat anti–rabbit immunoglobulin G antibody (Santa Cruz Biotechnology) at room temperature for 2 hours. Fluorescent images were captured by the FluoView laser scanning confocal microscope (Olympus).

Frozen sections were placed at room temperature for 30 minutes and then blocked and permeabilized in 5% bovine serum albumin with 0.3% Triton X‐100 (Sigma‐Aldrich) for 30 minutes at 37°C. Slides were incubated with primary antibody for collagen type I analysis (rabbit anti–collagen type I; Abcam), collagen type III analysis (rabbit anti–collagen type III; Sigma‐Aldrich), and transforming growth factor β1 (TGF‐β1) analysis (rabbit anti‐TGF‐β1; Santa Cruz Biotechnology) and mouse anti‐vimentin (Boster Bioengineering) overnight at 4°C and with secondary antibody (Santa Cruz Biotechnology) at room temperature for 1.5 hours. Fluorescent images were captured by the FluoView laser scanning confocal microscope (Olympus).