Plan apo 1.4na

Cells were grown on coverslips at 70–80% confluency. Cells were labeled with EdU (10 µM) for 15 min to visualize cells in S-phase. After 15 min, cells were washed and incubated in fresh medium containing HU (4 mM) for 3 h. After 3 h cells were washed in PBS and fixed in 2% paraformaldehyde in PBS containing 0.1% Triton-X and permeabilized for 15 min in 0.5% Triton-X in PBS. Coverslips were washed three times and subsequently stained with RIF1 primary antibody (1:5000, rabbit) for 1 h at RT. After 1 h, cells were washed with PBS containing 0.1% Triton-X followed by incubation in secondary antibody conjugated to Alexafluor-488 for 1 h at RT. EdU was visualized with a click-it reaction (click-it EdU imaging kit, Invitrogen) using a 594 nM fluorescent azide according to the manufacturer’s protocol. Coverslips were washed and incubated with DAPI (4',6-diamidino-2-phenylindole) for 10 min and mounted with ProLong Gold antifade reagent (Invitrogen). In experiments using ionizing radiations, cells were irradiated with (10 Gy) and allowed to recover for 2 h followed by 2% paraformaldehyde fixation and immunostaining. Images were obtained using Carl Zeiss Axio Imager D2 microscope using 63X Plan Apo 1.4 NA oil immersion objective and analyzed with ImageJ software64. Details of the antibodies are provided in Supplementary Tables 1 and 2.

Super-resolution structured-illumination microscopy images were obtained using a Zeiss Elyra PS.1 microscope (488 and 561 nm excitation wavelengths) with a × 63 Zeiss objective (Plan Apo 1.4NA oil immersion). The fluorescence signal was detected with an EMCCD camera (iXon-885, Andor). Centriolar proteins co-localization was assessed on egg chambers fixed with 4% PFA and mounted in Citifluor medium. Co-localization of the MUD and NUP107 proteins on the nuclear envelope was assessed ex vivo, in live egg chambers mounted in a Voltalef 10 s oil droplet. 0.17 mm high-performance Zeiss coverslides were used. Images were acquired for up to 25 μm in depth.

HAP1 cells were plated on 35-mm microscopy-grade plastic dishes (Ibidi). After dox induction and subsequent imposition of chemical ER stress, the cells were imaged over 8 h using a Marianas fluorescence microscope equipped with an OKO Lab CO₂ enclosure on a Zeiss inverted platform, with a 63X Plan-Apochromat 1.4 NA objective. Images were collected as in (23 (link)). Exposure times varied between 0.1 and 0.5 s, depending on sample intensity, unless otherwise specified. In some experiments, cells were imaged using a 63X Plan Apo 1.4 NA objective on an Axiovert 200M (Zeiss) with a spinning disc confocal system (UltraVIEW ERS6, PerkinElmer). Images were collected using an ORCA Flash 4.0 camera (Hamamatsu Photonics) using Volocity V.6.3.1 acquisition software.

Monitoring the expression and aggregation of SOD1-EGFP and SOD1^T54R-EGFP in IMR-32 cells were done by fluorescence microscopy. Cells were fixed by 4% paraformaldehyde (in PBS, pH: 7.4), followed by blocking of the cells with 1% bovine serum albumin (in PBS, pH: 7.4) and intermittent washing with PBS (pH: 7.4). Image acquisition was done in LSM700 (Zeiss) confocal laser scanning microscope using 63× Plan Apo/1.4 NA oil immersion objective. Image processing was done in Zen-lite (Zeiss) software. The overall process of cell preparation and microscopy was similar to one of our previous studies.^{57 (link)}