Hcx plapo 63 1.4 objective

Confocal images of the stained cells were obtained from a Leica TCS-SP5 microscope with a HCX PLAPO 63×/1.4 objective. Images were acquired with 300 Hz scanning speed in 15 μm image stacks with a 0.5 μm step size. For the time-lapse images of mitosis progression, HeLa-S3-tub-EYFP cells were seeded in chambered coverslips (μ-slide 4 well, ibidi GmbH, Germany) and imaged on a Leica TSC-SP5 inverted microscope with a HCX PLAPO 40×/0.85 objective, 30 μm image stack, 0.5 μm step size and 10 min imaging span under a 37 °C and 10% CO₂ environment. Laser intensity and HyD gain were fixed for each independent experiment, and the 3D confocal images were processed and projected into 2D with ImageJ. Quantitative analyses of confocal images were conducted with Imaris software. To determine the accumulation of HSP70 and other centrosome markers, a “Spot” of 1 or 2 μm in diameter surrounding the centrosome/spindle poles was created, and the intensity sum within the spot was measured. To determine the size of the centrosome, a “surface” covering the fluorescence signal of a centrosome marker was created, and the signal volume was measured. The conditions for immunostaining, imaging, and spot/surface creation were consistent for each independent experiment. Values for intensity and volume were normalized to markers that were stained and imaged in each experiment.

Cells seeded on coverslips were fixed, and immunofluorescence staining was performed as previously described^{69 (link)}. The primary antibodies included anti-α-tubulin (GTX112141, GeneTex, Hsinchu, Taiwan or T5168, Sigma), anti-γ-tubulin (T6557 or T3559, Sigma), anti-pericentrin (ab4448 or ab28144, Abcam, Cambridge, UK) and anti-TOMM20 (ab56783, Abcam). Alexa-Fluor 488-, 568-, 633-, and 647-conjugated goat anti-mouse, as well as anti-rabbit IgG were purchased from Invitrogen (Carlsbad, CA, USA). Confocal images of the immunostained samples were obtained with a Leica TCS-SP5 microscope equipped with a HCX PLAPO ×63/1.4 objective at 300 Hz scanning speed; image stacks of 15–20 μm were collected with a 0.5-μm step size. The confocal image stacks were processed and maxima-projected in ImageJ for presentation.