Quorum wavefx spinning disc confocal system

The iPSCs were seeded onto 12-well plates coated with Geltrex, grown to confluency, washed twice with PBS (−/−) and fixed with 10% formalin for 20 min at room temperature. The fixed cells were washed with PBS (−/−) then incubated with blocking buffer (10% FBS, 0.1% in PBS) for 30 min at room temperature to prevent any non-specific antibody binding. The primary antibodies were prepared by diluting with antibody buffer (0.2% FBS, 0.1% Triton X-100 in PBS stored at 4 °C). The following primary antibodies were used: Oct4 (1:100 dilution, Invitrogen), Sox2 (1:100 dilution, R&D Systems, USA) and Nanog (1:10 dilution, ReproCell, USA). After blocking, the wells were washed once with PBS (−/−) with 0.1% Triton X-100 and 300 µl of the primary antibody was added to each well. Primary antibodies were incubated overnight at 4 °C. After primary antibody incubation, the cells were washed (4 × 15 min) and the respective secondary antibodies were added to the cells and then incubated for 30 min at room temperature in the dark. Afterwards, the cells were washed (5 × 15 min) and stained with DAPI. All fluorescence images were taken on the spinning disk confocal (Quorum WaveFX Spinning Disc Confocal System). Secondary antibody controls were done to show no cross-reaction with the tissue (Figure S1).

Muscle sections were visualized with a spinning disk confocal microscope (Quorum Wave FX Spinning Disc Confocal System – Quorum technologies). Z-stacked images taken across a tissue section using the ×20/0.85 oil lens were assembled together and plane-merged to create a composite image to enable the visualization of a whole and clear tissue cross-section. Volocity 6.3 software [PerkinElmer, Waltham, MA, USA] was used to capture, merge, and analyze all images.

Transverse EDL muscle sections (10 μm) were cut (Leica Microsystems CM1850, Nussloch, Germany) in duplicate from the proximal and distal parts of the muscles. Tissue sections stained with hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO, USA) were examined with an inverted microscope (Nikon, Ontario, Canada) and damage, regenerating and intact areas on approximately 100 myofibers per muscle were quantified with ImageJ software version 1.41 (National Institutes of Health, USA). The damaged area was defined as an area not occupied by normal or regenerating muscle fibers. Image series of EDL were taken using a confocal microscope (Axio Observer.Z1; Carl Zeiss, Germany) and acquired using a Quorum WaveFX spinning disc confocal system (Quorum Technologies, Ontario, Canada). Solid state laser lines 491 nm and 561 nm were used for excitation of green and red (Alexa-488 and Alexa-594), combined with appropriate BrightLine single-bandpass emission filters (536/40 nm and 624/40 nm, Semrock, NY, USA). z-series were acquired at the same time with DAPI fluorescence filter cube (Chroma Technology, VT, USA). The CCD camera used to capture the images was a Hamamatsu Image EM C-9100. Images were acquired and analyzed using Volocity software, version 4.2.1. Iterative restoration (deconvolution) was applied for the DAPI channel, using the same software.