μ slide glass bottom slides

Forty-eight hours before confocal imaging, 60,000 cells were seeded in 8-well 15 μ-Slide glass bottom slides (Ibidi). PA (100 nM), LF_N-eGFP (200 nM), and PI (1 μg/mL) were added to the cells with fresh medium. The panel of cell lines was incubated using 50 nM PA. Cells were imaged 3 h after addition of components at 37°C and 5% CO₂ on a Nikon Eclipse Ti-E inverted microscope with a Yokogawa spinning disc system W1, a 100x oil objective, and an incubation system for live cell imaging including a stage-top incubator for chambered cover glass.

Immunocytochemistry was performed on 96-well plates (Perkin Elmer) or μ-Slide glass bottom slides (Ibidi). On day 15 post differentiation, cells were fixed using 4% paraformaldehyde for 15 minutes at room temperature. Following three PBS washes, cells were permeabilized using 0.1% Triton-X100 for 10 minutes at room temperature. Cells were then blocked in 2% bovine serum albumin for 60 minutes. Primary antibody incubation was performed overnight at 4°C at 1:500 concentration of anti-TDP-43 antibody (Abcam ab254166). Immunofluorescence was detected with fluorochrome-conjugated secondary antibodies CF640 donkey anti-mouse (1:1,000) for detection of TDP-43. Finally, nuclei were counterstained with Hoechst (2 μg/mL, Thermo Fisher Scientific no. 62248). Images were acquired on an inverted Nikon spinning-disk confocal microscope (Nikon Eclipse T1), using a 60× 1.40 NA oil-immersion objective. Twelve images were captured per well with four replicates per condition. Quantification of nuclear TDP signal was performed using CellProfiler. Total mean intensities of nuclear TDP-43 signal were calculated as well as means per well. Data was tested for normality using Shapiro-Wilk test and unpaired t-test was applied to well means to determine significance. Data was visualized in GraphPad Prism 9.5.1.