Cells were seeded on slides (2D culture) or on IBIDI glass bottom dishes (3D culture) and were cultured until desired confluency. Cells were fixed with 4% PFA in PBS+/+ (PBS with 0.9 mM CaCl2 and 0.5 mM MgCl2) for 15 min at room temperature for slide and 30 min for IBIDI. Immunofluorescence staining was performed as previously described 7. Confocal images were acquired with the Zeiss LSM 780 laser scanning confocal microscope using 40x Plan‐Apochromat objective (NA = 1.4) or with Olympus FluoView FV100 confocal microscope using 20x and 40x objective (NA = 0.75). Image acquisition software was ZEN (black edition, LSM 780; blue edition Cell Observer) and with Olympus FluoView viewer software, respectively.
Plan apochromat objective
Manufactured by Olympus
The Plan Apochromat objective is a high-performance microscope objective designed to provide excellent optical performance. It is characterized by its ability to minimize chromatic and spherical aberrations, resulting in sharp, high-contrast images across a wide field of view.
Automatically generated - may contain errors
Lab products found in correlation
Ixon 897, by Oxford Instruments (2 mentions)
Csu x1, by Yokogawa (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 780, by Olympus (1 mentions)
Fluoview viewer software, by Olympus (1 mentions)
Fluoview fv100 confocal microscope, by Olympus (1 mentions)
Andor neo zyla camera, by Oxford Instruments (1 mentions)
Nunc thermanox, by Thermo Fisher Scientific (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using plan apochromat objective
1
Immunofluorescence Imaging of 2D and 3D Cultures
2
Spinning-Disk Confocal Imaging of Fission Yeast
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We imaged cells on gelatin pads in growth chambers containing EMM5S medium and 100 µM n-propyl gallate at 25°C. We used an inverted microscope (Olympus, IX-71) with a 100×/1.4 numerical aperture Plan Apochromat objective (Olympus) fitted with a spinning-disk confocal head (CSU-X1; Yokogawa Corporation of America), electron-multiplying charge-coupled device camera (iXon 897; Andor Technology), argon-ion lasers (Melles Griot), acousto-optical tunable filters (Gooch and Housego), and dichroic mirrors and filters (Semrock). Images were acquired using Andor IQ2 software.
For still images, we took z-series of twenty-one 260-nm confocal slices encompassing 5.2 µm, which covered the entire cell. For time-lapse imaging, we took z-series of three 400-nm confocal slices closest to the coverslip at 1- or 2-s intervals for ∼200 s.
For still images, we took z-series of twenty-one 260-nm confocal slices encompassing 5.2 µm, which covered the entire cell. For time-lapse imaging, we took z-series of three 400-nm confocal slices closest to the coverslip at 1- or 2-s intervals for ∼200 s.
3
Imaging Cells in Growth Chambers
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We observed cells on gelatin pads in growth chambers of EMM5S media at 25°C on an inverted microscope (IX71; Olympus) with a 100×, 1.4 NA Plan Apochromat objective (Olympus), argon ion lasers (Melles Griot), a spinning-disk confocal head (CSU-X1; Yokogawa Corporation of America), and an electron-multiplying charge-coupled device camera (iXon 897; Andor Technology) using iQ2 acquisition software (Andor Technology). We imaged cells at 31 or 36°C on agar rather than gelatin pads. For time-lapse images, we took z series of 13–20 confocal slices at 300–400-nm intervals encompassing the entire cell or three to seven slices closest to the coverslip.
4
Immunofluorescence Staining and Quantification
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Cells were cultured on glass coverslips (Nunc Thermanox, ThermoFisher), then fixed at the end of each experiment for 45 min with 4% PFA at room temperature. After 2 washes in TBS, the cells were incubated with blocking buffer (TBS with 5% goat serum and 5 mg/ml BSA) at room temperature for 45 min. Samples were subsequently incubated with primary antibodies diluted in blocking buffer overnight at 4 °C with shaking. The next day, samples were washed three times in TBS and incubated with secondary antibodies diluted in blocking buffer for 2–4 h at room temperature. The cells were washed three times and the coverslips were then mounted on glass slides using Prolong Gold antifade reagent mounting medium with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI; Invitrogen). Samples were visualized using a Nikon Ti-E inverted fluorescence microscope equipped with Andor Neo/Zyla camera (Andor) and NIS elements advanced research software (version 4.2, Nikon) and a Plan Apochromat objective (Olympus). Fluorescence quantification was done by acquiring several images in different locations of each monolayer, and then measuring the integrated density (IntDen) of the appropriate channel using Fiji19 (link).
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