Affinipure fab fragment goat anti mouse igg

Antibodies used were anti-Pax7 (mouse IgG1, dilution 1:100, catalog No. AB_528428; Developmental Studies Hybridoma Bank, Iowa City, Iowa), antibody of laminin (rat or rabbit, dilution 1:1,500, catalog No. L0663 or L939; Sigma-Aldrich), anti-Ki67 (dilution 1:400, catalog No. 9129S; Cell Signaling Technology, Danvers, Massachusetts), anti-myogenin (F5D, dilution 1:100, sc-12732; Santa Cruz Bio-technology, Dallas, Texas), anti-GAPDH (dilution 1:2,500, ab9485; Abcam, Cambridge, Massachusetts), AffiniPure Fab fragment goat anti-mouse IgG (H + L, AFFGAI, 0.1 mg/ml; catalog No. 115–007-003; Jackson ImmunoResearch), AlexaFluor 594-conjugated goat anti-mouse IgG (H + L, dilution 1:1,500, catalog No. A-11032,; Life Technologies, Grand Island, New York), AlexaFluor 488-conjugated goat anti-mouse IgM (dilution 1:1,500, catalog No. A-21042; Life Technologies), and AlexaFluor 647-conjugated goat anti-rabbit or anti-mouse antibodies (dilution 1:1,500, catalog No. A-21244 or A-21235; Life Technologies). All reagents that are not specifically documented were purchased from Sigma-Aldrich.

10 μm sections from OCT-embedded QUAD muscles were cut onto glass slides using a microtome-cryostat. Sections were fixed with 4% paraformaldehyde for 20 min at room temperature, washed with phosphate-buffered saline (PBS), blocked with 10% normal goat serum (NGS) for 45 min, washed with PBS, blocked with AffiniPure Fab fragment goat anti-mouse IgG (1.3 mg/ml in PBS; 115–007–003; Jackson ImmunoResearch, West Grove, PA, USA) for 30 min, washed with PBS, then incubated at 4 °C overnight in mouse anti-laminin IgG2a primary antibody (1:100 dilution in 10% NGS; D18; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; deposited by Sanes, J.R.). Sections were then washed with PBS, incubated in goat-anti mouse IgG2a 546 secondary antibody (1:500 dilution in 10% NGS; #A21133; Thermo Fisher Scientific, Waltham, MA, USA) and 10 μg/ml DAPI for 45 min, washed with PBS and mounted with a glass coverslip and Fluormount-G (Southern Biotech, Birmingham, AL, USA). 5–7 Images were acquired at 20× magnification using fluorescence microscopy and average fiber area of a total of at least 600 fibers per muscle was measured using Image J software (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/download.html).