One day before the assay L4 males were isolated and allowed to mature to adulthood on MitoTracker Red (MTR) containing plates. Plates were prepared by pipetting a dilution of 5 μL of 1mM MTR in 50 μL of M9 buffer onto the OP50 lawn and allowing it to dry. Males were grown overnight at 20°C in the dark. 1 hour before mating, males were moved to a recovery plate without MTR. Hermaphrodites were immobilised (0.1% tetramisole for 5–10 min) and then added to mating plates with ∼50 males for 30 min). Half of the hermaphrodites were immediately imaged every 30 sec on a Nikon C2 Basic Confocal microscope with a 100X oil immersion objective for sperm tracking analysis. The other half of the hermaphrodites were imaged 1 hr after mating for zone localisation analysis. The experiment was repeated twice. Tracking analysis and zone localisation analysis was performed as described in Hu et al.24 (link) Briefly, sperm were tracked using the TrackMate plugin in ImageJ.85 (link) The uterus was divided into thirds (zone 1 closest to the vulva, zone 3 closest to the spermatheca) and only those sperm visible in zone 2 were tracked. The same zones were used to classify sperm distribution throughout the uterus in the 1 hr post-mating images. Only animals where sperm were distinct enough to count were classified.
C2 basic confocal microscope
Manufactured by Nikon
The Nikon C2 Basic Confocal Microscope is a compact and cost-effective laser scanning confocal imaging system designed for a wide range of applications. It features a small footprint, simplified operation, and high-quality imaging capabilities.
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2 protocols using c2 basic confocal microscope
1
Sperm Tracking and Localization in C. elegans
2
Imaging Caenorhabditis elegans Fluorescence
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Animals were scored for the presence or absence of GFP with a Nikon SMZ18 Microscope.
For Figure 3A-E and Figure S5A, animals were immobilised in M9 with 0.2% Tetramizol and mounted on glass slides. DIC and fluorescent imaging was performed using standard methods using a Leica Sp5 Multiphoton confocal microscope (SET-32::mCherry and mCherry::SET-25) and a Nikon Ti-E spinning disk microscope (mCherry::SET-25). For SET-32::mCherry analysis, all images of the tagged strain and N2 control animals were taken under identical conditions.
For immunofluorescence experiments in Figure 3F-H and Figure S5B-D, germlines were imaged using a Nikon C2 Basic Confocal microscope. Within conditions (DAPI, H3K9me2, and H3K9me3 staining) all strains were imaged under identical parameters.
For Figure 5E-G, synchronized L4 hermaphrodites were incubated for 12 or 24 hours, then fixed as previously described (Nishimura et al., 2015) . DNA was visualized with DAPI. Imaging was performed using an Olympus FluoView FV1000 Confocal microscope, and processed using Olympus FluoView software.
For Figure 3A-E and Figure S5A, animals were immobilised in M9 with 0.2% Tetramizol and mounted on glass slides. DIC and fluorescent imaging was performed using standard methods using a Leica Sp5 Multiphoton confocal microscope (SET-32::mCherry and mCherry::SET-25) and a Nikon Ti-E spinning disk microscope (mCherry::SET-25). For SET-32::mCherry analysis, all images of the tagged strain and N2 control animals were taken under identical conditions.
For immunofluorescence experiments in Figure 3F-H and Figure S5B-D, germlines were imaged using a Nikon C2 Basic Confocal microscope. Within conditions (DAPI, H3K9me2, and H3K9me3 staining) all strains were imaged under identical parameters.
For Figure 5E-G, synchronized L4 hermaphrodites were incubated for 12 or 24 hours, then fixed as previously described (Nishimura et al., 2015) . DNA was visualized with DAPI. Imaging was performed using an Olympus FluoView FV1000 Confocal microscope, and processed using Olympus FluoView software.
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