Imaris x64

Microscopic analysis of the NP adherence experiments in the 96-well microtiter plates was performed with the More™ fluorescence microscope system (FEI Life Sciences, formerly TILL Photonics, Gräfelfing, Germany) using the following settings: Oligochrome light source, objective lens 10×, filter set ET480/40×, T495LPXR, ET535/50m (FITC). Single wells were scanned by applying the Relative Tile experiment setting where a tile of 5 by 6 images around a selected position is acquired. The individual tiles were fused to one relative tile by the Live Acquisition Software (version 2.22, FEI Life Sciences Munich GmbH, Germany). The total fluorescence intensity in the relative tile was determined by ImageJ (version 1.46a, NIH, Bethesda, USA). The statistical analysis was performed with Graph Pad Prism (version 5.04, Graph Pad Software, Inc., La Jolla, USA).
The cells on the transwell filters were microscopically examined with the same settings as above, except for the objective lens which was changed to 40× and the additional use of a second filter set (ET620/60x, T660LPXR, ET700/75m; Cy5). With both filter sets (FITC and Cy5) z-stacks were recorded over 20 frames with 1 µm z-slices by using Live Acquisition Software. After processing the 2D image sets in ImageJ the 3D rendition was performed with Imaris x64 (version 7.4, Bitplane AG, Zurich, Switzerland).

Specimens were analyzed and light micrographs were taken on an Olympus BX53 microscope equipped with an Olympus DP73 camera and the software cellSens Standard, Version 1.11 (Olympus Corporation, Shinjuku, Tokyo, Japan). Confocal laser scanning microscopy was conducted using a Leica DMI6000 CFS microscope equipped with a Leica TCS SP5 II scanning system (Leica Microsystems, Wetzlar, Germany) and software LAS AF, Version 2.6.0 or 2.6.3. The autofluorescent signal was scanned in fluorescence mode using a 405 nm laser and gene expression signal was scanned in reflection mode using a 633nm laser (see [44 (link)–47 (link)]). The obtained confocal image stacks were processed and used to prepare 3D reconstructions of the gene expression signal as well as 3D renderings of larval tissue with Imaris x64, Version 7.3.1 (Bitplane AG, Zurich, Switzerland). The same software was used to create video files of larval stages including gene expression patterns. Generated images were finally processed with Adobe Photoshop CS6 Extended, Version 13.0.1 x64, and Adobe Photoshop CC 2015 (Adobe Systems). Figures and schematic drawings were generated with Adobe Illustrator CS5, Version 15.0.0 and Adobe Illustrator CC 2015, Version 1.0 (Adobe Systems).

Cells were cultured on a φ35-mm glass-bottom dish, and time-lapse imaging was performed every 15 min using a BioStation IMQ (Nikon, Tokyo, Japan), which is a small incubation unit equipped with a humidified imaging chamber set at 37 °C, 5% CO₂ in air and with a microscope. Time-lapse images were processed by Imaris x64 (ver. 7.6.5.; Bitplane AG, Zurich, Switzerland).

For sterile muscle injury, mice were anesthetized with isoflurane and 50 μL cardiotoxin (12 × 10^–6 M, 217503, Millipore) injected in the TA muscle. Muscles 8 days after injury were snap frozen in nitrogen-chilled isopentane. 8 μm cryosections were cut and stained with H&E. For each analysis, more than 10 slides (per condition/group) containing 6 muscle sections/sample were used, and myofibers in the injured area were counted and measured with a Mirax digital scanner and HALO software (68 (link)). For adult mouse studies, muscles were fixed in 10% formalin and embedded in paraffin for sectioning. Laminin staining (RB-082, Thermo Fisher Scientific) was used to outline fibers, and picrosirius red was used for collagen accumulation. Image analysis was performed using ImarisX64 software (Bitplane AG), and fiber sizing was calculated using minimal Feret’s diameter (69 (link)). For after weaning juvenile mouse studies, cryosections from soleus muscles were mounted in ProLong antifade mountant with DAPI (Thermo Fisher Scientific). Fiber type, size, and central nucleation were quantified with analysis via SMASH (70 (link)) and ImageJ (NIH). Antibodies used were mouse anti-MHC2A (1:50, SC-71, Developmental Studies Hybridoma Bank) and mouse anti-eMHC (1:10, BF-45, Developmental Studies Hybridoma Bank).