Confocal microscopy, fluorescence microscopy, and EM

Confocal images were captured as serial sections along the Z-axis on a Zeiss LSM710 confocal microscope with a 63×/1.4 NA oil objective. The acquired images were processed with deconvolution (regularized inverse filter) by ZEN software. Then, the sharp regions from the individual sections of Z-stack images were combined to form extended depth of focus single images. The focus of all the z-positions were calculated to one extended depth of focus image, which enabled the display of a considerably larger depth of field than is possible with a single focus. For live-cell imaging using a Zeiss LSM710 confocal microscope (related to Fig. 4, E, G, and H), cells transfected with GFP-tagged TDP-43 or mutations were maintained on a stage-top incubator (37°C, 5% CO₂). Fluorescent signaling was monitored at 5-s intervals. All images were processed using Zen software and Fiji (ImageJ).

Fluorescence images from brain sections, cultured neurons, and HEK293 cells were captured with a Zeiss Celldiscoverer 7 automated microscope (controlled by Zen software; Zeiss). For live-cell time-lapse imaging, HEK293 cells were seeded into a 35-mm glass-bottomed dish and transfected 24 h after seeding using Lipofectamine 2000 with 0.5 µg GFP-TDP-43, GFP-TDP-43ΔCR, GFP-TDP-43ΔNLS, and GFP-TDP-43ΔNLSΔCR constructs. 24 h after transfection, the culture dish was placed into the Celldiscoverer 7 system with a stage-top incubator (37°C, 5% CO₂). All the images were captured by the water immersion objective at 100× magnification.

For transmission EM, samples were freshly dissected and processed. Small pieces of cortical tissue were fixed by immersion in triple-aldehyde–DMSO. After being rinsed in distilled water, they were postfixed in ferrocyanide-reduced osmium tetroxide. Another water rinse was followed by an overnight soak in acidified uranyl acetate. After being rinsed in distilled water again, the tissue blocks were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in Poly/Bed resin. Thin sections were sequentially stained with acidified uranyl acetate followed by a modification of Sato’s triple-lead stain and examined in an FEI Tecnai Spirit (T12) with a Gatan US4000 4k × 4k charge-coupled device. All EM images were obtained and quantified blindly and independently by investigators without knowledge of the samples.