Las ax software

Unless otherwise specified, all microscopy images were acquired with a Leica DM6000 microscope equipped with a DFC390 camera and LAS AX software (Leica) at 63× magnification. During acquisition, fields were selected randomly in DAPI staining except for the scoring of MiDAS and mitotic aberrations, where fields containing mitosis were specifically acquired. The minimum number of cells estimated for analysis were 50 mitosis or 300 cells for PLA and IFs and 100 cells for comet assays. Intensity values and number of foci were scored automatically with Metamorph v7.5.1.0 software to avoid bias.

All images were acquired using a Leica SP5 confocal microscope and analyzed offline using the Leica LASAX software from z-stack images as described previously (Mentis et al. 2011 (link); Fletcher et al. 2017 (link)). Images for motor neuron number quantification were acquired using a 20× objective at 3-µm steps in the Z-axis. Images for VGluT1⁺ inputs were acquired using a 40× objective at 0.3-µm steps in the Z-axis. Images for NMJ innervation quantification were obtained using a 20× objective at 2-µm steps in the Z-axis. Images for SmB intensity analysis were acquired using a 40× objective at identical settings for wild-type and SMA samples at 3-µm steps, and analysis was performed using LASAX software by measuring the peak intensity per unit area of the nuclear region of motor neurons.