Wide‐field images for qualitative or quantitative analysis were acquired using a Zeiss Z2 Axioimager microscope with MetaSystems VSlide slide scanning software (20× dry magnification lens, 0.9 NA) with a Colibri 7 solid‐state fluorescent light source. Filters were described and validated previously [33 (link)]. Exposure times for each channel were set per case because of differences in immunoreactivity; UBQLN2‐linked ALS+FTD case MN17 showed poor immunoreactivity overall, likely because of long‐term fixation, therefore longer exposures were used. Confocal images were acquired using an Olympus FV1000 confocal microscope (100× magnification oil immersion lens, 1.4 NA, Z‐step 0.4–1 μm) with FluoView 4.2 software (Olympus). Maximum intensity Z‐projections were generated and processed using FIJI software (v1.53c, National Institutes of Health).
Final figures were compiled using FIJI and Adobe Illustrator (Adobe Systems Incorporated, v24.3). Extracted single‐channel images were imported into FIJI, merged and pseudocoloured. For each channel, image intensity was adjusted in FIJI to best view the marker of interest, reduce background autofluorescence and generate images representative of ubiquilin 2 and TDP‐43 aggregate burden in each region.
Final figures were compiled using FIJI and Adobe Illustrator (Adobe Systems Incorporated, v24.3). Extracted single‐channel images were imported into FIJI, merged and pseudocoloured. For each channel, image intensity was adjusted in FIJI to best view the marker of interest, reduce background autofluorescence and generate images representative of ubiquilin 2 and TDP‐43 aggregate burden in each region.
