Picrosirius Red Staining of Immobilized Muscle

Cryosections of vastus lateralis muscle from the immobilized limb after 14 days of post-immobilization recovery were stained for collagen expression using picrosirius red (PSR) staining, as described previously [28 (link)]. Fifteen-micrometer sections of muscle were fixed in ice-cold acetone, washed in PBS, and stained in Bouin’s reagent (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The sections were then rinsed in water, stained with PSR (Sigma Direct Red 80, Sigma-Aldrich) for 1 h, and rinsed in acidic water (5 mL glacial acetic acid in 1 L of water) and a picric alcohol (10% picric acid, 20% ethanol) rinse. The slides were then cleared in xylene and mounted with Permount medium (Sigma-Aldrich). Slides were imaged in a blinded manner using an Olympus DP72 Digital Camera System mounted to an Olympus BX51 TRF Microscope (Olympus, Center Valley, PA, USA), and the collagen fiber area was determined using custom image analysis software (programmed by RGB; full code can be accessed at https://github.com/rbudnar/PSR).