Ab34360

A portion of the neotissue from the defect region was embedded in a talcum-based gel, frozen in 2-methylbutane, and supercooled in liquid nitrogen. Cryosections (8 μm) were prepared and probed for laminin (1:100; catalog ab34360; Abcam) and GFP (1:100; catalog ab6673; Abcam) and detected with fluorescent antibodies (1:200; catalog A11055 and A21207; Invitrogen) to assess direct myogenic contribution of BMNCs. Qualitative assessments were made by observing three sections (separated by no less than 160 μm) from five muscles per group.

Protein extracts were separated in 12% SDS–gel electrophoresis, using antibodies against p62 (dilution 1 μg/ml, Abcam^®, ab56416) and pax7 (dilution 1 μg/ml, Abcam^®, ab34360). Beta‐tubulin antibody (dilution 1:1,000, Cell Signaling Technology^®, #2128) was used as a loading control, and membranes were imaged using Bio‐Rad ChemiDoc^™ XRS+ with Image Lab software.

A portion of the TA muscle from the defect region was embedded in a talcum-based gel, frozen in 2-methylbutane, and supercooled in liquid nitrogen. Cryosections (8 μm) were prepared and stained using standard protocols for hematoxylin & eosin. For immunofluorescence, sections were probed for laminin (1:100; catalog ab34360; Abcam), GFP (1:100; catalog ab6673; Abcam), CD68 (1:50; MCA341A488; BioRad), and Pax7 (5 μg/mL; AB_528428; Developmental Studies Hybridoma Bank) and detected with fluorescent secondary antibodies (1:200; catalog A11055, A21207, and A32723; Invitrogen). Brightfield and fluorescent images were acquired with a Zeiss Axio Scan.Z1 and stitched into a composite image depicting the cross section of the TA muscle belly. Qualitative assessments of morphology and composition were made by observing three sections from 5 muscles per group. Quantitative analyses were performed on the indexed image values after global thresholding and segmentation in MATLAB (Mathworks, Natick, MA, USA). For regional analyses, segmented images where multiplied with a binary mask representing each of the defect region and the remaining musculature.

The proximal half of the TA muscle belly (inclusive of the defect region) was embedded in a talcum-based gel, frozen in 2-methylbutane, and supercooled in liquid nitrogen. Cryosections (8 μm) were prepared and stained using standard protocols for hematoxylin & eosin and Masson’s trichrome. For immunofluorescence, sections were probed for laminin (1:100; catalog ab34360; Abcam) and either GFP (1:100; catalog ab6673; Abcam), CD68 (1:50; MCA341A488; BioRad), or CD3 (1:50, catalog ab185763; Abcam). Positive staining was detected with fluorescent secondary antibodies (1:200; catalog A11055, A21202, and A21207; Invitrogen). Brightfield and fluorescent images were acquired with a Zeiss Axio Scan.Z1 and stitched into a composite image depicting the cross section of the TA muscle belly. Qualitative assessments of morphology and composition were made by observing three sections from 5 muscles per group. Quantitative Analyses were performed on the indexed image values after global thresholding and segmentation in MATLAB (Mathworks, Natick, MA, USA). Defect regions were defined based on visual inspection and qualitative assessments of myofiber size and organization. Remaining muscle mass was defined as the remainder of the section not included in the defect region.