For DIC and fluorescence microscopy imaging of spermatheca dynamics, day 1 adult hermaphrodites with one row of embryos were mounted on glass slides with 10% agarose pads, which served to immobilize them, and 1.38 µl of M9 medium. Image acquisition was performed at 20°C on a Ti-Eclipse inverted microscope (Nikon) equipped with a CSU-X1 spinning-disk confocal head (Yokogawa Electric Corporation), DPSS-Laser (Roper Scientific) of 491- and 561-nm excitation wavelengths and an Evolve electron multiplying charged-coupled device camera (Photometrics). Focus drift during time-lapse acquisition was corrected using Perfect Focus System (Nikon). Acquisition control was performed using MetaMorph (Molecular Devices). Ovulation and embryo transit movies were acquired with a 60× 1.4 NA oil-immersion Plan-Apochromat objective (Nikon). Live super-resolution imaging of protein localization in the spermatheca (Fig. 6, A and B ; and Fig. S5 ) was performed on a Ti-2 Eclipse with a Live-SR module (Gataca systems, France) mounted on the light path of a CSU-W1 spinning disk, a 100× 1.45 NA oil-immersion Plan-Apochromat objective (Nikon, Japan), and a Prime95B SCMOS camera (Photometrics). Actin stress fibers in the spermatheca (Fig. 2 H and Fig. 7 A ) and embryos images (Fig. 1 B and Fig. 5 C ) were acquired with a 100× 1.45 NA oil-immersion Plan-Apochromat objective (Nikon).
Plan apochromat oil immersion objective
Manufactured by Nikon
Sourced in Japan
The Plan Apochromat oil-immersion objective from Nikon is a high-performance microscope lens designed for advanced imaging applications. It features a precise, aberration-corrected optical design that provides excellent image quality and resolution across the entire field of view.
Automatically generated - may contain errors
Lab products found in correlation
Csu x1 spinning disk confocal head, by Yokogawa (1 mentions)
Perfect focus system, by Nikon (1 mentions)
Dpss laser, by Teledyne (1 mentions)
Csu w1 spinning disk, by Nikon (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Zyla 4.2 scmos camera, by Oxford Instruments (1 mentions)
H3k27me3, by Thermo Fisher Scientific (1 mentions)
Ti e inverted microscope, by Nikon (1 mentions)
Phospho h3s10, by Abcam (1 mentions)
H3k9me2, by Cell Signaling Technology (1 mentions)
Fibrillarin, by Abcam (1 mentions)
H3k27ac, by Active Motif (1 mentions)
H3k9ac, by Cell Signaling Technology (1 mentions)
Csu 22 confocal head, by Yokogawa (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Eclipse c1, by Nikon (1 mentions)
4 protocols using plan apochromat oil immersion objective
1
Imaging Spermatheca Dynamics in C. elegans
2
Immunofluorescence Imaging of Chromatin and Nuclear Proteins
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Cells were fixed and prepared for indirect immunofluorescence and confocal microscopy as previously described11 ,26 (link).
Images were acquired on a Nikon Ti-E inverted microscope (Nikon) with a Yokogawa CSU-22 spinning disk confocal head with the Borealis modification or a Ti2 inverted microscope fitted with a CSU-W1 spinning disk. Z-stacks of 0.4–0.7 μm spacing were collected using a CoolSnap HQ2 CCD camera (Photometrics) or a Zyla 4.2 sCMOS camera (Andor) with a ×60/1.40 NA or a ×100/1.45 NA Plan Apochromat oil-immersion objective (Nikon).
The following antibodies were used for indirect immunofluorescence imaging: phospho γH2AX (Ser139) (Millipore, 05-636-I; 1:400); H3K27ac (Active Motif, 39133; 1:200); MDC1 (Abcam, ab11171; 1:1,000); MDC1 (Sigma-Aldrich, M2444; 1:1,000); phospho RNA PolII S5 (Millipore, MABE954, clone 1H4B6; 1:400); Cdk9 (Cell Signaling, 2316; 1:10); CDK12 (Abcam, ab246887; 1:400); 53BP1 (Santa Cruz, 22760S; 1:100); H3K27me3 (ThermoFisher Scientific, MA511198; 1:1,000); H3K9ac (Cell Signaling, 9649S; 1:400); H3K9me2 (Cell Signaling, 9753S; 1:400); POM121 (Proteintech, 15645-1-AP; 1:200); phospho H3T3 (Millipore, 07-424, 1:12,000); phospho H3S10 (Abcam, ab47297; 1:200); and fibrillarin (Abcam, ab4566; 1:500). Staining of Dam-methylated DNA in fixed cells was done using purified GFP-tagged m6A-Tracer protein as previously described53 (link).
Images were acquired on a Nikon Ti-E inverted microscope (Nikon) with a Yokogawa CSU-22 spinning disk confocal head with the Borealis modification or a Ti2 inverted microscope fitted with a CSU-W1 spinning disk. Z-stacks of 0.4–0.7 μm spacing were collected using a CoolSnap HQ2 CCD camera (Photometrics) or a Zyla 4.2 sCMOS camera (Andor) with a ×60/1.40 NA or a ×100/1.45 NA Plan Apochromat oil-immersion objective (Nikon).
The following antibodies were used for indirect immunofluorescence imaging: phospho γH2AX (Ser139) (Millipore, 05-636-I; 1:400); H3K27ac (Active Motif, 39133; 1:200); MDC1 (Abcam, ab11171; 1:1,000); MDC1 (Sigma-Aldrich, M2444; 1:1,000); phospho RNA PolII S5 (Millipore, MABE954, clone 1H4B6; 1:400); Cdk9 (Cell Signaling, 2316; 1:10); CDK12 (Abcam, ab246887; 1:400); 53BP1 (Santa Cruz, 22760S; 1:100); H3K27me3 (ThermoFisher Scientific, MA511198; 1:1,000); H3K9ac (Cell Signaling, 9649S; 1:400); H3K9me2 (Cell Signaling, 9753S; 1:400); POM121 (Proteintech, 15645-1-AP; 1:200); phospho H3T3 (Millipore, 07-424, 1:12,000); phospho H3S10 (Abcam, ab47297; 1:200); and fibrillarin (Abcam, ab4566; 1:500). Staining of Dam-methylated DNA in fixed cells was done using purified GFP-tagged m6A-Tracer protein as previously described53 (link).
3
Spinning Disk Confocal Microscopy Imaging
4
Endocytosis Inhibition in Cell Samples
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After encapsulation, cell suspension (0.2 mL) was centrifuged for 10 min at 5100×g and 25 °C and 0.2 mL of water-glycerol solution at 15.0 MPa and 4 °C was added to the cells to stop natural endocytosis (Dupont et al. 2010; (link)Marechal et al. 1995; (link)Pedrini et al. 2014 (link)). The samples were protected from light and kept in ice during analysis. A Nikon Eclipse TE 2000 U microscope (Nikon, Tokyo, Japan) with multispectral confocal head D Eclipse C1 was used to observe cells stained with fisetin. Excitation was performed at 488 nm with laser He/Ar, and the emission signal was recovered between 490 and 670 nm. Images were acquired with a ×100 (NA: 1.4) Plan
Apochromat oil-immersion objective (Nikon) and collected with NIS software 3.1 (Nikon).
Apochromat oil-immersion objective (Nikon) and collected with NIS software 3.1 (Nikon).
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