Immunohistochemistry was performed as described previously [11 (link)]. The rabbit anti-BrdU (1:200; Rockland, Philadelphia, Pennsylvania, USA) was used and detected in 3,3′-diaminobenzidine. Slides were counterstained with hematoxylin. The percentage of BrdU-positive cells was calculated by counting the total number of basal cells of granulation tissues and cells expressing nuclear BrdU stain.
Immunofluorescence was also performed as described previously [12 (link)]. The rabbit anti-BrdU (1:200; Rockland) or mouse monoclonal anti-α-smooth muscle actin antibody (1:300; Sigma) was used and detected with secondary donkey anti-mouse or rabbit IgG-Cy3 antibody (1:200; Beyotime). Cells were counterstained with the nuclear dye 4′,6-diamidino-2-phenylindole (Beyotime) and examined with a fluorescence microscope.
Sirius red staining was performed as described previously [11 (link)]. In brief, paraffin-embedded sections were dehydrated and stained in Sirius red solution for 1 hour, then mounted with Poly-Mount Xylene. Polaroid lens were used for images taken under a microscope.
Immunofluorescence was also performed as described previously [12 (link)]. The rabbit anti-BrdU (1:200; Rockland) or mouse monoclonal anti-α-smooth muscle actin antibody (1:300; Sigma) was used and detected with secondary donkey anti-mouse or rabbit IgG-Cy3 antibody (1:200; Beyotime). Cells were counterstained with the nuclear dye 4′,6-diamidino-2-phenylindole (Beyotime) and examined with a fluorescence microscope.
Sirius red staining was performed as described previously [11 (link)]. In brief, paraffin-embedded sections were dehydrated and stained in Sirius red solution for 1 hour, then mounted with Poly-Mount Xylene. Polaroid lens were used for images taken under a microscope.