Image acquisition

Samples labeled with fluorescent dyes were imaged in a dark room. Images were acquired using the same acquisition settings across all samples for each immunohistochemical label. Slides were imaged using a Nikon Eclipse upright fluorescent microscope equipped with a Prior Scientific XY motorized stage and a Zyla 4.2 PLUS monochrome camera (Andor), and cells were imaged using a Nikon Eclipse Ti2 inverted fluorescent microscope. Nikon NIS-Elements software was used for image acquisition and XY stitching. Images were captured with a 10× or 20× magnification objective. Confocal microscope was performed using an Olympus FV1000. To generate representative images (not used for quantification), the Extended Depth of Focus module in NIS-Elements was sometimes used to create focused images from Z-stacks. Images were exported as 8-bit TIFF files for analysis. For NMJ imaging, images were acquired using a Zeiss LSM 780 confocal system or a Leica DMR epifluorescence microscope equipped with a Hamamatsu cooled CCD camera controlled by a Macintosh computer with iVision software (BioVision Technologies).