Softworx suite

Cells were grown on glass coverslips and fixed for 8 min in ice-cold MeOH at −20°C. Cells were blocked in blocking solution (2.5% FBS, 200 mM glycine, 0.1% Triton X-100 in PBS) for 1 h at room temperature or overnight at 4°C. Cells were then incubated in primary antibody diluted in the blocking solution for 1 h and rinsed with PBST (0.1% Triton X-100 in PBS), followed by secondary antibody staining prepared in the same blocking solution. DNA was stained with DAPI, and cells were mounted in ProLong Gold antifade mountant (Invitrogen). The following primary antibodies were used: rabbit centrin (1:1000; in-house, raised against human centrin2 [amino acids 1–172]) and goat CEP192 (1:1000; in-house, raised against CEP192 [amino acids 1–211]). Both primary antibodies were directly conjugated to Alexa fluor 488, 555, or 647 (Thermo Fisher Scientific). Details on antibodies are also listed in Supplemental Table S1.
Immunofluorescence images were acquired at room temperature (25°C) using a DeltaVision Elite system (GE Healthcare) controlling a scientific CMOS camera (pco.edge 5.5). Images were acquired using an Olympus 40×, 1.35 NA oil objective with Applied Precision immersion oil (n = 1.516) at 40× with 0.2-µm z-sections. Acquisition parameters were controlled by SoftWoRx suite (GE Healthcare).

Immunofluorescence images were acquired using a DeltaVision Elite system (GE Healthcare) controlling a scientific CMOS camera (pco.edge 5.5). Images were acquired with Applied Precision immersion oil (n = 1.516) at 100X or 60X with 0.2 μm z-sections. Acquisition parameters were controlled by SoftWoRx suite (GE Healthcare). U-ExM images of MEFs, RPE1, and DLD1 cells were acquired using a Leica SP8 confocal microscope with a 63X/1.4 NA oil immersion objective with 0.2 µm z-step size and a pixel size of 22.55nm. Immunofluorescence and U-ExM MCC images were acquired using a Leica SP8 confocal microscope with a 40X plan-apochromat oil immersion objective with 1.30 NA, or a Zeiss Axio Observer 7 inverted microscope with Slidebook 2023 software (3i—Intelligent, Imaging Innovations, Inc.), CSU-W1 (Yokogawa) T1 Super-Resolution SoRa Spinning Disk, and Prime 95B CMOS camera (Teledyne Photometrics) with a 40X plan-apochromat oil immersion objective with 1.30 NA. Images were deconvolved with Leica’s lightning process software or Microvolution software built into Slidebook. Images were processed in FIJI (Schindelin et al., 2012). All images presented in figures are Z-stack maximum intensity projections. All imaging analysis was performed blinded using ImageJ (v2.1.0/ 1.53c, National Institutes of Health, http://imagej.net). Statistical analysis was performed using GraphPad Prism software.