M165 fluorescent dissecting microscope

Live worms, colorimetric WISH stains, and whole-worm H3P stains were imaged on a Leica M165 fluorescent dissecting microscope. dFISH stains were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head. H3P cell counts were quantified using freely available ImageJ software (http://rsb.info.nih.gov/ij/) with the cell counter function, and muscle lesions were labelled by hand, using control RNAi animals as a reference so as to only label muscle gaps larger than those found in controls, and quantified using the ImageJ area measurement function. Piwi-1⁺ and H3P⁺ cell counts were performed in cross-section using Imaris (Bitplane, South Windsor, CT, USA). Cross-sections were taken from the same regions in control and knockdown animals (examples shown in Fig 3A), and “normal” and “ectopic” regions were determined manually using DAPI staining to mark the boundary (as shown in Fig 3B). Collagen⁺ cells, TUNEL⁺ cells, and neoblast subclass markers were also quantified using Imaris. Significance was determined by a two-tailed unequal variance student’s t-test. All images were post-processed in a similar manner using Adobe Photoshop. Heatmaps were made in R studio using data downloaded from https://compgen.bio.ub.edu/PlanNET/planexp [61 (link)].

Colorimetric whole-mount in situ hybridization stains were imaged on a Leica M165 fluorescent dissecting microscope. Fluorescent stains were imaged on a spinning disk confocal microscope (Olympus IX81S1F-3) with a Hamamatsu C9100-13 EM-CCD camera and a Yokogawa CSU X1 scan head, employing Perkin Elmer Volocity software. All image quantifications and post-processing were made using the freely available ImageJ software Fiji (http://rsb.info.nih.gov/ij/) and Imaris (http://www.bitplane.com/imaris/imaris). All experiments were, at minimum, triplicated and at least 10 worms were used per stain and per time point (i.e. n≥30). All statistical analyses between RNAi groups were carried out using a two-tailed Student's t-test. All images were post-processed using Adobe Photoshop and figures assembled in Inkscape.