Computational protocol: Pervasive satellite cell contribution to uninjured adult muscle fibers

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Protocol publication

[…] Myofiber images were captured on a Leica DMRXA upright spinning disc confocal microscope. Objectives were 10×/0.3 NA HC Plan Fluotar, 20×/0.7 NA HC Plan Apo, or 40×/0.85 NA HC X Plan Apo (correction collar). Leica was equipped with a Yokagawa CSU10B spinning disk, and images were taken with a Hamamatsu ImagEM EM-CCD. Muscle sections were imaged on either the Leica or Zeiss 510 LSM. Objectives used on the Zeiss were 10×/0.3 NA EC Plan Neofluar, 20×/0.8 NA Plan Apo Chromat, or 63×/1.4 NA oil differential interference contrast Plan Apo Chromat M27 lens (Carl Zeiss). Images were processed using MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices) or the FIJI ImageJ version 1.47 package (NIH) with the additional MacMaster BioPhotonics Facility plugin set. Confocal stacks were projected as maximum intensity images for each channel, background subtracted, and merged into a single image in ImageJ. Brightness and contrast were adjusted for the entire image as necessary. Images were either cropped or merged as necessary, and individual color channels were extracted without color correction or ɣ-adjustment. Images were adjusted and counted manually. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus