Dechorionated ctB>UAS-CD8-GFP embryos were mounted on double-sided Scotch tape in PBS solution. The TC and surrounding two or three cells were ablated (to ensure removal of both TC and SC) in late 12/early 13 stage embryos. Cell ablation was performed using a 63×/0.9 NA water immersion lens on a Yokogawa spinning disk (CSU-10) confocal microscope fitted with a pulsed nitrogen laser (MicroPoint). Image acquisition and microscope control were by MetaMorph (version 7.0) software (Molecular Devices). Embryos were allowed to develop to stage 16 under humid conditions at 25°C, fixed and processed for immunostaining.
Metamorph version 7.0
Manufactured by Molecular Devices
MetaMorph (version 7.0) is a software package for image acquisition, processing, and analysis. It provides a comprehensive suite of tools for researchers to capture, enhance, and quantify digital images from a variety of microscopy techniques.
Automatically generated - may contain errors
3 protocols using metamorph version 7.0
1
Ablation of Trichome Cells in Drosophila Embryos
2
Yeast Endosomal Membrane Staining
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Liquid cultures of yeast strains were grown to logarithmic phase at 30°C before observation at room temperature using a Nikon TE2000-U inverted fluorescence microscope equipped with a Yokogawa CSU-Xm2 spinning-disk confocal unit and a 100× oil objective with a numerical aperture of 1.4 (Nikon Instruments, Melville, NY). Fluorescence images were acquired with a Photometrics (Tucson, AZ) Cascade II electron-multiplying CCD camera using MetaMorph (version 7.0) software (Molecular Devices, Sunnyvale, CA) and then processed with ImageJ (National Institutes of Health, Bethesda, MD) and Photoshop CS4 software (Adobe Systems, Mountain View, CA). Endosomal membranes were stained with FM 4-64 (Invitrogen, Carlsbad, CA) using a 20-min pulse and 90-min chase (Odorizzi et al., 2003 (link)).
3
Fluorescence Microscopy of GFP and FM 4-64
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An inverted microscope (IX81; Olympus) equipped with an UPLANSAPO 100× objective lens (numerical aperture 1.40; Olympus) and a charge-coupled device camera (DP30BW; Olympus) was used for fluorescence microscopy. The filter sets used to acquire fluorescence signals were GFP-4050B (Semrock) for GFP and a U-MRFPHQ unit (Olympus) for FM 4–64 signals. Acquired images were processed using MetaMorph version 7.0 (Molecular Devices).
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