Histological and immunohistochemical analyses
Tissue samples were fixed in 10% formalin neutral buffer solution for 24 h, and then embedded in paraffin before being cut into 4 µm-thick sections.
For staining of collagen (Masson’s trichrome staining), sections were treated sequentially with primary mordant agent, Weigert’s iron haematoxylin, secondary mordant agent, 0.75% Orange G solution, Masson’s staining solution B, 2.5% phosphotungstic acid solution, and finally with aniline blue solution. Samples were mounted with malinol, and inspected with a fluorescence microscope (Olympus DP71) or scanned using a NanoZoomer-XR digital slide scanner. Image J software (National Institutes of Health, Bethesda, MD) was used to calculate the percentage of collagen positive area.
For immunohistochemical analysis of α-SMA, sections were blocked with 2.5% goat serum for 10 min, incubated for 12 h with an antibody against α-SMA (1:100 dilution) in the presence of 2.5% bovine serum albumin, and then incubated with Alexa Fluor 594 goat anti-rabbit immunoglobulin G (1:500 dilution) and DAPI (5 µg/ml) for 1 h. Samples were mounted with VECTASHIELD and inspected with a fluorescence microscope (Olympus DP71). Image J software (National Institutes of Health, Bethesda, MD) was used to calculate the percentage of α-SMA positive area.
For histological examination, sections were stained first with Mayer’s haematoxylin and then with 1% eosin alcohol solution (H&E staining). Samples were mounted with malinol and inspected with a fluorescence microscope (Olympus DP71).