haematoxylin and eosin (Sigma-Aldrich) staining was performed by 90 seconds of incubation in haematoxylin followed by 30 seconds of incubation in eosin. Trichrome Masson staining samples were fixed with Bouin’s solution (Sigma-Aldrich) for 15 min at 56°C followed by Wiegerts iron haematoxylin (Sigma-Aldrich) for 5 min, then a Trichrome kit (Sigma-Aldrich) and finally incubated with 1% acetic acid for 2 min. A bright field microscope was used to take 10x & 20x images. Periodic Acid-Schiff reagent (Sigma-Aldrich) was used to preform PAS staining following standard protocols.
Immunofluorescence analyses of cell count were visualized with DAPI (Vector Laboratories Burlingame). Minimum of 60 10x bright field microscopic images (Leica DM5500 B Microscope System) per experimental group were counted for positive expression of DAPI. ImageJ software determined cell count. Immunofluorescence analysis was conducted by 1hr incubation with primary antibodies collagen IVα1/α2, collagen I, fibronectin, PCNA and Bcl-associated X protein (Bax), followed by a 30 min incubation with secondary anti-mouse or anti-rabbit Alexa Fluor 555 (Life Technologies Grand Island, NY, 1:500) SeeSupplementary Table 2 for antibody dilution. DAPI mounting (Vector Laboratories) was used to visualize samples with a Leica DM5500 B Microscope System.
Immunofluorescence analyses of cell count were visualized with DAPI (Vector Laboratories Burlingame). Minimum of 60 10x bright field microscopic images (Leica DM5500 B Microscope System) per experimental group were counted for positive expression of DAPI. ImageJ software determined cell count. Immunofluorescence analysis was conducted by 1hr incubation with primary antibodies collagen IVα1/α2, collagen I, fibronectin, PCNA and Bcl-associated X protein (Bax), followed by a 30 min incubation with secondary anti-mouse or anti-rabbit Alexa Fluor 555 (Life Technologies Grand Island, NY, 1:500) See