Nagata et al. Supplementary Figure S1

Supplemental Figure S1. Representative images of inducible nitric oxide synthase-expressing (iNOS+) cells on postinjury day 5. A-B: Images were obtained from the damaged muscle in the icing group. A: A transverse section of injured muscle was analyzed by immunohistochemical staining for iNOS, counterstained with hematoxylin. Black arrowheads indicate iNOS+ regenerating muscle cells with central nuclei. B: A transverse section of injured muscle was analyzed by immunofluorescence for CD68 (green) and iNOS (red). Nuclei were stained with 4′-6-diamino-2-phenylindole (DAPI, blue). Arrows indicate macrophages identified as positive color of CD68 surrounding a nucleus (blue). White arrowheads indicate iNOS+ cells excluding macrophages identified as single positive color of iNOS surrounding a nucleus (blue). Scale bars = 30 μm. Method for (A): Immunohistochemical staining was performed on the cross section of injured extensor digitorum longus muscle. Cryosection was fixed with 4% paraformaldehyde, washed in phosphate-buffered saline (PBS), blocked and permeabilized with PBS containing 10% normal goat serum at room temperature for 1 h, and incubated with primary antibodies at 4°C overnight. The primary antibody: rabbit polyclonal anti-iNOS antibody (1:50, ab3523, lot# GR3382908-1; Abcam, Cambridge, UK; RRID: AB_303872) was diluted in PBS containing 5% normal goat serum. After washing the section three times in PBS, endogenous peroxidase activity was quenched with 0.3 % hydrogen peroxide diluted in methanol for 30 min at room temperature. The section was then washed three times in PBS and was incubated with MAX-PO (R) (Nichirei Biosciences Inc., Tokyo, Japan) secondary antibodies for 30 min at room temperature. After washing with PBS, the section was placed in the 3,3′-diaminobenzidine reaction solution, counterstained with hematoxylin. The sample was viewed on an Olympus BX50 microscope (Olympus, Tokyo, Japan) with objectives and image was taken with a digital camera (DP20; Olympus). Method for (B): Immunofluorescence was performed on the cross section of injured extensor digitorum longus muscle. Cryosection was fixed with 4% paraformaldehyde, washed in PBS, blocked and permeabilized with PBS containing 10% normal goat serum and 1% Triton X-100 at room temperature for 1 h, and incubated with primary antibodies at 4°C overnight or at room temperature for 1 h. The primary antibodies: mouse monoclonal anti-CD68 antibody (1:200, MCA341GA, lot# 0515; Bio-Rad, Hercules, CA, USA; RRID: AB_566872), and rabbit polyclonal anti-iNOS antibody (1:50, ab3523, lot# GR3382908-1; Abcam, Cambridge, UK; RRID: AB_303872) were diluted in PBS containing 5% normal goat serum. After washing the section three times in PBS, the sample was incubated with the appropriate species-specific (anti-mouse and/or anti-rabbit) fluorescence-conjugated (Alexa Fluor 488 and/or 594) secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA), diluted (1:1000) in PBS containing 5% normal goat serum, at room temperature for 2 h. After washing with PBS, the section was mounted using Vectashield mounting medium (Vector Laboratories, Burlington, ON, Canada) containing 4′-6-diamino-2-phenylindole to visualize the nuclei. The sample was viewed for the fluorescent labels on a ZEISS Axio Vert. A1 microscope (Zeiss, Oberkochen, Germany) and photographed with an AxioCam MRm (Zeiss) camera (max resolution 1388 × 1040 pixels) using AxioVision Rel. 4.8 (Zeiss) software.

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