Fluorescence imaging was performed as described previously (Kowalski et al., 2011 ). Briefly, animals were immobilized using 30 mg/ml 2,3-butanedione monoxamine (Sigma-Aldrich) and the anterior ventral nerve cord was imaged. 1μm (total depth) Z-series stacks were collected using a Carl Zeiss Axiovert M1 microscope with a 100× Plan Apochromat (1.4 numerical aperture) objective equipped with GFP and red fluorescent protein filters. Images were collected with an Orca-ER CCD camera (Hamamatsu) and MetaMorph (version 7.1) software (Molecular Devices). Maximum intensity projections of Z-series stacks were used for quantitative analyses of fluorescent puncta. Exposure settings and gain were adjusted to fill the 12-bit dynamic range without saturation and were identical for all images taken of each fluorescent marker. Line scans of ventral cord puncta were generated using Meta-Morph (version 6.0) and were analyzed with Igor Pro (version 5) (Wavemetrics) (Burbea et al., 2002 (link)). Arc lamp output was monitored by measuring the fluorescence intensity of 0.5 μm FluoSphere beads (Invitrogen).
Metamorph version 7.1
Manufactured by Molecular Devices
Sourced in United States
MetaMorph (version 7.1) is a software application designed for comprehensive image acquisition, processing, and analysis. It provides a suite of tools for controlling microscope hardware, capturing high-quality images, and performing quantitative measurements on biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Orca er ccd camera, by Hamamatsu Photonics (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Fluosphere beads, by Thermo Fisher Scientific (1 mentions)
Axiovert m1 microscope, by Zeiss (1 mentions)
Meta morph, by Wavemetrics (1 mentions)
2 3 butanedione monoxamine, by Merck Group (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
2 protocols using metamorph version 7.1
1
Fluorescence Imaging of Ventral Nerve Cord
2
Immunofluorescence Analysis of H4K20me3
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Cells grown on glass coverslips were fixed and permeabilized for 10 minutes with 4% paraformaldehyde containing 0.5% Triton X-100, and then blocked with 5% BSA. Next, cells were incubated with a primary antibody against H4K20me3 and appropriate secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, UK). Finally, coverslips were mounted in ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Fluorescence intensity was analyzed using a fluorescence microscope (IX81; Olympus, Tokyo, Japan). The staining intensities were measured using MetaMorph version 7.1 (Molecular Devices, Sunnyvale, CA, USA).
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