Picrosirius red stain kit

Mouse aorta samples were isolated, fixed with 10% formaldehyde in PBS at room temperature for 24 h, embedded in paraffin, and sectioned at 7 μm. Aorta morphological histomorphometric characters were analyzed via hematoxylin/eosin (H&E) staining. Picrosirius red stain kit (Polysciences Inc. Warrington, PA, USA) was performed to identify collagen fibers, following the manufacturer’s instructions. H&E staining and Picrosirius red-stained collagen were observed under light microscopy (Leica Imaging Systems, Cambridge, UK). The digital photomicrographs were quantified with Image-Pro Plus Software (Media Cybernetics, USA). The stainings were assessed and quantified blindly by two independent observers.

Sections of TA muscles were harvested at five or 14 days post-injury from adult or aged Ctl and P7:S4KO mice. Regenerated skeletal muscles harvested five days after injury were immunostained with embryonic Myosin Heavy Chain (eMyHC) antibodies to label actively regenerating fibers and average cross-sectional area of 250–300 myofibers was quantified with ImageJ. Regenerated skeletal muscles harvested 14 days after injury were processed for H and E or Sirius Red staining. For H and E, flash-frozen sections were fixed for 3 min in 4% PFA, stained with Mayers Hematoxylin and Alcoholic Eosin Y, dehydrated, equilibrated with xylene and mounted using Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI). For Sirius Red staining, a Picrosirius Red stain kit (Polysciences, Warrington, PA) was utilized. Frozen sections were fixed for 1 hr at 56°C in Bouin’s fixative, washed in water, stained for 1 hr in Picrosirius Red, washed in 1 M HCl, dehydrated, equilibrated and mounted. Bright-field images were collected with a Zeiss Axioskop 40 microscope. To obtain quantification of average cross-sectional area and frequency distribution of 14dpi regenerated fiber size, Myosin/Laminin immunostained TA sections were analyzed using ImageJ software.

Immunohistochemical staining was carried out as described previously [28 (link)]. A mouse anti-Human ACLP/AEBP1 mAb (1:100 dilution, LS-C133036; LSBio), mouse anti-α-smooth muscle antigen (α-SMA) mAb (1:50 dilution, M0851; CiteAb, Bath, UK), and mouse anti-CD8 mAb (clone C8/144B; DAKO) were used. Areas positive for ACLP and α-SMA were measured using ImageJ software ver. 1.52 (NIH, Bethesda, MD, USA) as described [29 (link)]. Collagen I was stained using a Picrosirius Red Stain Kit (Polysciences, Warrington, PA, USA) according to the manufacturer’s instructions.