HeLa cells (1 × 105) were seeded onto glass coverslips in a 12-well plate and treated as indicated. Cells were fixed with 4% PFA for 15 min, permeabilized with 0.2% Triton X-100/TBS/3% BSA for 20 min, and blocked with 0.2% Tween-20/TBS/3% BSA for 1 hour at room temperature. Primary antibodies were diluted in 0.2% Tween-20/TBS/3% BSA and incubated overnight at 4°C. Secondary antibodies were also diluted in 0.2% Tween-20/TBS/3% BSA and incubated at room temperature for 1 hour. Coverslips were mounted with mounting medium containing DAPI (Abcam, ab104139) and visualized by confocal fluorescence microscopy (Zeiss LSM 800 confocal laser scanning microscope with a Plan-Apochromat 40×/1.4 oil differential interference contrast M27 objective). Representative images were chosen out of two independent experiments.
Plan apochromat 40 1.4 oil
Manufactured by Zeiss
The Plan-Apochromat 40×/1.4 oil is a high-performance microscope objective lens produced by Zeiss. It features a magnification of 40x and a numerical aperture of 1.4, which provides a high level of optical resolution and image quality. The lens is designed for use with oil immersion, which allows for increased light-gathering capability and improved contrast in microscopy applications.
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3 protocols using plan apochromat 40 1.4 oil
1
Immunofluorescence Imaging of HeLa Cells
2
Steady-state Fluorescence Kinetics of Anabaena Heterocysts
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Steady-state fluorescence kinetics of WT and ΔnblA heterocysts of Anabaena 33047 were measured by the fluorescence kinetic microscope (Photon Systems Instruments, Drasov, Czech Republic, www.psi.cz ). PBS was excited with green LED light (LZ1-00G100, 530 nm peak). A steady-state PBS fluorescence was separated from exciting light by a dichroic mirror with 562 nm edge wavelength (Semrock 562 nm edge BrightLine) and collected using an emission filter with transmission band 663.5 to 666.5 nm (Semrock 660/13 nm BrightLine). Signals were measured from individual heterocysts and vegetative cells using the 40× objective (Zeiss Plan-Apochromat 40×/1.4 oil). The average signal from a minimum of 10 cells was used to obtain the representative PBS signal from the mutant and the WT. The FluorCam7 software developed by Photon Systems Instruments was used to operate the FKM and analyze the data.
3
Sperm Visualization in C. elegans
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Adult Pcomp-1::GFP::H2B::3’comp-1 animals 24 h post-L4 lethargus were sealed into individual “artificial dirt” chambers filled with NGM and 10 μM levamisole. Confocal images of fluorescently labeled sperm were obtained using a Zeiss LSM 880 microscope with a Plan-Apochromat 40×/1.4 oil differential interference contrast (DIC) objective. Image stacks were analyzed using the FARSIGHT Nucleus Editor (http://www.farsight-toolkit.org/wiki/NucleusEditor ). We note that when the fluorescent marker was crossed to a daf-16 mutant background, it was not confined to the nuclei. However, individual sperm cells were still detectable. We observed this in all daf-16(mgDf60) mutants and 25% of the daf-16(mu86) mutants.
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