Openlab software

We collected the culture progeny of MuSCs from aged Myf5^nLacZ/+/Luciferase double-transgenic mice^{23 (link),43 (link)} by incubation with 0.1% trypsin in PBS for 2 min at 37 °C and transplanted them into tibialis anterior muscles of hindlimb-irradiated NOD/SCID mice. One month after transplant, we injected notexin to damage recipient muscles and activate MuSCs in vivo. Four days later, we collected, fixed, and cryosectioned recipient muscles, as described above. We performed immunohistological analysis of transverse tissue sections to detect β-galactosidase⁺ cells (indicating a donor-derived cell expressing Myf5, a marker of MuSC activation) in the satellite cell position within the myofiber basal lamina, as defined by laminin staining. We stained sections with anti-Laminin (Millipore, clone A5, catalog # 05-206, 1:250) and anti-β-galactosidase (Invitrogen, catalog # A11132, 1:100) primary antibodies and then with appropriate secondary antibodies (Invitrogen). We counter-stained nuclei with Hoechst 33342 (Invitrogen). We acquired images with an AxioPlan2 epi-fluorescent microscope (Carl Zeiss) with Plan NeoFluar 10×/0.30NA or 20×/0.75NA objectives (Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu). We captured digital images in OpenLab software (Improvision) and assembled them using Photoshop software (Adobe) with consistent contrast adjustments across all images.

Mice were euthanized and lungs were removed 6 hours after tail vein injection of GFP labeled tumor cells (5×10⁵) together with Gr-1+CD11b+ (1.5×10⁶) or RAW264.7 cells (2×10⁵). Images were normalized to the basal signal obtained from lungs 1 hour after tail vein injection. Lungs were observed under the fluorescent microscope, 10 random pictures under 10X magnification were taken and further analyzed as fluorescence signal per field by OpenLab software (Improvision) or ImageJ. For extravasation experiment, mice were injected with 100ul of 12.5ug/ul 70,000MW tetramethylrhodamine (Invitrogen). Lungs were imaged under Zeiss 510 NLO confocal microscope. Images were analyzed with Zeiss ZEN software.

Sciatic nerves distal to crush injury sites from scLRP1^−/− and littermate control mice were processed for plastic embedding as described previously (Orita et al., 2013 (link)). Briefly, glutaraldehyde (2.5%)/paraformaldehyde (2%)/0.1 M sodium phosphate, 150 mM NaCl, pH 7.4 (Fixation Buffer) was applied directly onto mouse sciatic nerves prior to removal. Resected tissue was further incubated in Fixation Buffer for an additional 72 h at 4°C, washed, chilled in 0.1 M cacodylate Buffer, pH 7.4, secondarily fixed in 1% osmium tetroxide/CB for 30 min, dehydrated in serially increasing concentrations of ethanol and embedded in Durcupan resin (Sigma-Aldrich). Semi-thin sections (50–60 nm) were applied to copper grids (300 mesh). The grids were viewed using a Tecnai G² Spirit BioTWIN transmission electron microscope (TEM) equipped with an Eagle 4k HS digital camera (FEI, Hilsboro, OR) or with a JOEL FX1200 Transmission Electron Microscope. Quantitative TEM image analysis was performed. C-fibers in Remak bundles (number of Remak bundles, SC cytoplasmic abnormalities) and myelinated fibers (fiber number, wide incisures, lamellae, myelin outfoldings, myelin infoldings) were counted in a 500 μm² surface area of scLRP1^−/− and scLRP1^+/+ mice (3–5 sections per individual mouse). Nerves were imaged at 1900X and analysed using OpenLab software (Improvision, Conventry, UK).