Picrosirius Red Staining of Muscle Tissue

Picrosirius red staining was done similarly to other studies [14 (link), 15 (link), 38 , 39 (link)]. Before sectioning, fixed muscles were embedded in 4% agarose. 200 μm thick longitudinal sections were sliced using Leica VT1000S. Sections were washed, dried for 60 min, and stained for 60 min in 0.1% (weight/volume) Direct Red 80 (Fisher) dissolved in saturated aqueous picric acid (Fisher). Sections were washed twice for 60 s in 0.5% acetic acid, then dehydrated with three 60 s washes of 100% ethanol. The sections were then cleared using CitriSolv (Fisher Scientific) for 3 min, and blotted with Permount (Fisher Scientific).
Full longitudinal sections were imaged via a 20X objective with brightfield illumination on a Leica DMi8 microscope and DFC9000GTC camera. Linearly polarized light imaging required a rotating polarizer in the beam path before and after the sample. A sequence of ten tiling scans were imaged at angles from 0 to 90° in increments of 10°. ECM architecture was quantified using a custom MATLAB script providing MicroECM alignment and MacroECM deviation parameters as described previously [14 (link)] in conjunction with the polarized light images.