Weigert s hematoxylin staining reagent

For Masson’s trichrome staining, tissue cryosections were fixed with Bouin’s solution overnight, rinsed with water, and incubated with Weigert’s Hematoxylin staining reagent (Electron Microscopy Sciences) for 5 min. Slides were washed with water for 10 min and stained with a commercially available kit (Sigma Aldrich). Briefly, slides were first stained with Biebrich Scarlet-Acid Fuchsin Solution (Sigma Aldrich) for 5 min, rinsed in three changes of water, followed by staining with phosphotungstic/phosphomolybdic acid solution (Sigma Aldrich) for 10 min. Slides were then stained with aniline blue solution (Sigma Aldrich) for 5min to stain collagen in blue, differentiated with 1% glacial acetic acid for 2min, and then dehydrated with an ascending series of ethyl alcohol followed by xylene before they were mounted. Sections were imaged under the same conditions and intensities using a Nikon Ni widefield epifluorescence microscope with an objective lens magnification at 20×. Analysis of fibrotic area was conducted in Fiji software, using color deconvolution and thresholding function to define the areas of collagen and the muscle fiber. The fibrotic area percentage is calculated as (collagen area/muscle fiber area) × 100.