Plan apochromat 63 1.40 oil dic m27

Cells were grown on glass cover slips in 12-well plates before fixed with 4% paraformaldehyde. Permeabilized with 0.1% Triton X-100 for 15 min, and blocked with 10% normal goat serum for 1 hour. The coverslips incubation with the primary antibody at a 1:100 dilution overnight at 4°C. FITC-or Alexa fluor-labeled anti-rabbit or anti-mouse antibody was added to the incubation. The nuclei were stained with DAPI (Beyotime). The samples were observed under ZEISS LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany) and all images were captured using a 63×oil immersion objective (Plan-Apochromat 63×/1.40 Oil DIC M27).

For localization studies, cells were seeded on coverslips, fixed with 3% PFA (Sigma-Aldrich) or methanol, permeabilized with 0.05% saponin in PBS and stained with antibodies. In CHMP6 overexpression experiments, cells were permeabilized with PEM buffer (80 mM K-Pipes, pH 6.8, 5 mM EGTA, and 1 mM MgCl₂) before fixation.
Stained coverslips were viewed with an LSM 710 or 780 confocal microscope (Carl Zeiss) equipped with an Ar-laser multiline (458/488/514 nm), a DPSS-561 10 (561 nm), a laser diode 405–30 CW (405 nm), and an HeNe laser (633 nm). The objective used was a Plan-Apochromat 63×/1.40 oil DIC M27 (Carl Zeiss). Image processing was performed with basic software ZEN 2009 (Carl Zeiss) and ImageJ software (National Institutes of Health). CHMP4B and CHMP4C intensities at the intercellular bridge were scored manually using constant settings for the relevant channels. For CHMP6, EAP20 and α-tubulin intensity assessments values of gray intensities from 16-bit images were measured in a line along the intercellular bridge and plotted relative to the distance from the midbody identified by lack of tubulin staining. The mean intensity along the line (control set to 1) was compared between different treatments. The SEM is calculated between individual experiments.