Sp8 dmi8 confocal microscope

Cells were cultured on a coverslip before being fixed for 20 min using 4% PFA and rinsed three times with DPBS. Blocking was performed at room temperature for 1h using 5% normal donkey serum (NDS, Sigma), and 0.1% Triton X-100 in DPBS. Primary antibodies were diluted in 2% NDS, and 0.1% Triton X-100 in DPBS and incubated overnight at 4°C. A list of all antibodies used and their dilution can be found in Additional file 2: Table S1. After three washes with DPBS, the cells were incubated with the appropriate Alexa Fluor™ secondary antibodies (1:2000 in DPBS, Life Technologies; Additional file 2: Table S1) for 2h at room temperature. Subsequently, the coverslips were washed twice, and cell nuclei were stained using the NucBlue Live ReadyProbes™ Reagent (Thermo Fisher Scientific) for 20 min. Coverslips were again washed three times with DPBS before being mounted on microscope slides using ProLong™ Gold antifade reagent (Invitrogen). Confocal images were acquired using a Leica SP8 DMI8 confocal microscope (20 × or 64 × objective) using excitation lines at 405, 488, 555 and 647 nm. The acquired images were processed using Fiji software.

To assess colocalization between GR aggregates and KIF5B in C9-ALS/FTD postmortem tissues, we made use of brain and spinal cord tissue previously collected in the UZ Leuven brain biobank in accordance with the ethics review board upon written informed consent. In this study, five C9orf72 expansion carriers were used for the analysis in the frontal cortex of the brain and one C9orf72 expansion carrier was used for the analysis in the spinal cord (for clinical information, see table S1). ALS patients without the C9orf72 expansion were chosen as controls (table S1). Frozen tissue sections of 7 to 8 μm thickness were washed with Bond Wash Solution (Leica Biosystems) for 15 min at room temperature and stained overnight with the primary antibodies at 4°C (table S7). The following day, slides were washed three times with Bond Wash Solution before the secondary antibodies (table S7) were added for 90 min at room temperature. After three washing steps with Bond Wash Solution, the slices were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen). Confocal images were obtained using a Leica SP8 DMI8 confocal microscope. Images were analyzed, formatted, and quantified with ImageJ software.

Coverslips were incubated for 20 min at room temperature in PBS with 4% paraformaldehyde and washed three times with PBS. Cells were incubated for 1 hour at room temperature in PBS containing 0.1% Triton X-100 (Acros Organics) and 5% donkey (Sigma-Aldrich) or goat (Dako) serum. Cells were incubated with primary antibodies diluted in PBS containing 0.1% Triton X-100 and 2% serum overnight at 4°C. Cells were rinsed three times in PBS before the secondary antibody was added for 1 hour at room temperature. See table S7 for details of primary and secondary antibodies. After washing with PBS, the coverslips were incubated for 20 min in PBS/4′,6-diamidino-2-phenylindole (DAPI) (NucBlue Live ReadyProbes Reagent, Invitrogen). Coverslips were rinsed three times with PBS before mounting in ProLong Gold antifade reagent (Invitrogen). Confocal images were obtained with a Leica SP8 DMI8 confocal microscope. Images were analyzed, formatted, and quantified with ImageJ software.