Raw images and image processing scripts to reproduce results of the manuscript entitled 'Human and mouse ALS tissue sections immunolabeled for FUS, SFPQ, ChAT and counterstained with DAPI' and posted on BioRxiv in August 2020. 1. RowMouseImages.zip Tissue sections from spinal cord of SOD1- and VCP-mutant ALS mouse models together with control immunolabeled for FUS, SFPQ, ChAT and counterstained with DAPI. 2. HumanMouseImages.zip Tissue sections from spinal cord of Healthy and sporadic ALS donors immunolabeled for FUS or SFPQ, ChAT and counterstained with DAPI. 3. ImageProcessingScripts.zip Scripts to preprocess these images. 4. Data.zip CSV files of the single-cell measurements obtained from CellProfiler after segmentation as presented in ImageProcessingScripts.zip. Downstream analysis leading to automatic identification of MNs subpopulation can be acessed on Github https://github.com/RLuisier/ALSdisMNs --------- The two series of multichannel fluorescent microscopy data have been used in several manuscripts including: Luisier R, Tyzack GE, Hall CE, Mitchell JS, Devine H, Taha DM, et al. Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS. Nat Commun 2018; 9: 2010. Tyzack GE, Luisier R, Taha DM, Neeves J, Modic M, Mitchell JS, et al. Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis. Brain 2019; 142: 2572–80.