One-day-old adult worms were microirradiated on live-imaging slides in indicated zone (TZ (Figure 4 ) or MP (Figure S3 )) and then recovered. Ethanol fixation was performed with the following protocol at indicated time points following microirradiation for each worm (as indicated in Results , Figure 4 and Figure S3 ): 5–10 adult worms were placed on an uncharged slide (Surgipath Leica) in 5μl M9 solution, most liquid was soaked up using filter paper, then 5μl ethanol was applied and allowed to evaporate, followed by application of 5μl M9-DAPI dilution. Most of this solution was soaked up using filter paper again and a coverslip with Vectashield was placed on top. Coverslips were sealed with acrylic nail polish. Images were taken on the DeltaVision wide-field fluorescence microscope (GE Lifesciences) with 100x/1.4 NA oil Olympus objective. Images were then deconvolved with softWoRx software (Applied Precision). Microirradiation was indicated by the presence of bright green foci, as no foci appear at endogenous DSBs, and these nuclei were scored.
Deltavision wide field fluorescence microscope
Manufactured by GE Healthcare
The DeltaVision wide-field fluorescence microscope is a laboratory equipment designed for high-resolution imaging of fluorescently labeled samples. It utilizes an optical system to capture detailed images of cellular structures and dynamics.
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8 protocols using deltavision wide field fluorescence microscope
1
Ethanol Fixation for Microirradiated Worms
2
Acridine Orange Staining of Nuclei
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Acridine orange staining was performed on 1-day-old adults. Worms were transferred to a tube of 10 mg/ml acridine orange diluted 1:400 in M9, immediately covered with foil, and allowed to rotate on a mixer for 2 h. After incubation, these worms were transferred to a clean NGM plate. Worms were then placed onto a 10% agarose pad with 8 μl of M9 and 2 μl of Polybead 0.1-μm polystyrene beads (#00876; Polysciences), before imaging on the DeltaVision wide-field fluorescence microscope at 60× magnification (GE lifesciences). Levels of acridine orange-stained nuclei were quantified using softWoRx software (Applied Precision), where nuclei that stain positive were counted towards the total. Example images are shown in Supplementary Figure S3D .
3
Quantification of GFP::RPA-1 in C. elegans Gonads
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gfp::rpa-1 adult worms were grown to 1, 2, 3, and 4 days old. Their gonads were dissected on charged slides, frozen at −80°C, and placed in −20°C EtOH for 1 min then mounted with 10 μl M9-DAPI and VectaShield. Whole intact gonads were imaged on the DeltaVision wide-field fluorescence microscope (GE Lifesciences) in seven 512 × 512 pixel zones with 100x/1.4 NA oil Olympus objective from the proximal pre-meiotic tip to the distal late pachytene phase of prophase I. Each image was the center slice of the nuclei in the upper section of the syncytial tube in both the DAPI (blue) and FITC (GFP) fluorescent channels. Measurement of the intensity of each nucleus was taken in FIJI in each zone in the FITC channel and corrected to cytoplasmic background.
4
Quantifying Chromosome Cohesion Dynamics
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Cells carrying the GFP-LacI repressor and an array of Lac operators either next to the centromere of chromosome II to measure cohesion at the centromere or at position 1.95 Mb of the chromosome I to measure cohesion of a chromosome arm were fixed with 70% ethanol. Images were acquired using a DeltaVision wide-field fluorescence microscope (GE Healthcare). z-stacks with 15 images at 0.1 μm intervals were acquired and merged by maximum intensity projection. Quantification of the percentage of cells showing two separated GFP foci was performed using Fiji.
5
Cryosectioning and Immunostaining of Spleen
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Spleens were frozen in OCT compound on dry-ice, and sectioned on the cryostat (7-10µm thick). Sections were air dried overnight, then fixed in ice cold acetone for 15 mins at 4°C. Tissue sections were rehydrated in PBS for 10, and blocked in 5% NRS. Sections were stained in 5% NRS at 4°C overnight in a humidified chamber to detect IgM and MOMA-1, or CD1d, IgD and MOMA-1. Slides were washed PBS, and mounted in ProLong Gold antifade reagent (Thermo Fischer). Images were acquired at x10 or ×20 magnification using a DeltaVision widefield fluorescence microscope (GE Healthcare). Images were quantified using Image-J software (NIH). MZ width was measured using Image-J software. At least 10 follicles per genotype were imaged, measurements were taken from the edge of MOMA-1+ cells to the edge of CD1d+ staining. A number of measurements were taken per follicle to account for variability. CD1d MFI (-background) was calculated using Image-J (NIH). Follicular area was defined using MOMA-1 (CD169) staining. MFI was then calculated for this area (see Supplementary Fig. 8e ). MZ area was defined as CD1d+ area outside MOMA-1 and IgD staining (see Supplementary Fig. 8e ). Background fluorescence was calculated and subtracted from MFI values for MZ and FO. Measurements were taken from 10 follicles from 2 ERT2cre/+ chimeras, and 25 follicles from 2 Zfp36l1fl/flERT2cre/+ chimeras.
6
Cryosectioning and Immunostaining of Spleen
7
Live Imaging of Polystyrene Beads in Worms
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For live images, worms were placed on a slide with a 14% agarose in M9 pad and covered with 5ul of a 1:2 mixture of Polybead 0.1-μm polystyrene beads (#00876; Polysciences) in M9. The worms were covered with a coverslip and imaged. All images were taken using the DeltaVision wide-field fluorescence microscope (GE lifesciences) with 100×/1.4 NA oil Olympus objective. Images were deconvolved with softWoRx software (Applied Precision) unless otherwise noted.
8
Microirradiation and Ethanol Fixation of Adult Worms
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One day old adult worms were microirradiated on live imaging slides in indicated zone (TZ (Figure 4) or MP (Figure S3)) and then recovered. Ethanol fixation was performed with the following protocol at indicated time points following microirradiation for each worm (as indicated in Results, Figure 4 and Figure S3): 5-10 adult worms were placed on an uncharged slide (Surgipath Leica) in 5µl M9 solution, most liquid was soaked up using filter paper, then 5µl ethanol was applied and allowed to evaporate, followed by application of 5µl M9-DAPI solution. Most of this solution was soaked up using filter paper again, and a coverslip with Vectashield was placed on top. Coverslips were sealed with nail polish. Images were taken on the DeltaVision wide-field fluorescence microscope (GE Lifesciences) with 100x/1.4 NA oil Olympus objective. Images were then deconvolved with softWoRx software (Applied Precision). Microirradiation was indicated by the presence of bright green foci, as no foci appear at endogenous DSBs, and these nuclei were scored.
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