Ni widefield epifluorescence microscope

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.

Tibialis Anterior muscles were dissected and fixed in 4% PFA/PBS and incubated overnight in 30% sucrose prior to being embedded in OCT medium (Richard Allen Scientific) and frozen in chilled 2-methylbutane. Cryosections were cut at a thickness of 10 μm and placed on Superfrost plus slides. Sections were stained with Hematoxylin and Eosin. More specifically, slides were stained with Hematoxylin for 1min, rinsed in water, and incubated with acidic alcohol (1% hydrochloric acid in 100% ethanol). After rinsing in water, slides were immersed in bluing, water, and 70% ethanol for 30sec each. Next, slides were exposed to eosin for 1min and gradually dehydrated with a graduated ethanol series. After a final incubation in xylene, coverslips were mounted with Cytoseal-xyl. Sections were imaged on a Nikon Ni widefield epifluorescence microscope. Muscle fiber areas were manually determined using ImageJ/Fiji.