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This is a technique for intracellular labeling of small cells at gastrula stages or later.
Electronics
The electronics set-up is used as a means to get the dye out of the electrode and into the cell. Use an agar bridge (see Agar bridged ground wires, page 5.10) for the bath ground.
Pipettes
Success in single cell labeling lies primarily with the ability to make good pipettes. Pipettes should be pulled the day of the experiment. Allow at least 10 minutes for the tips to fill with dye. Electrodes are pulled from glass capillaries (e.g. Sutter thick wall, O.D. 1.2 mm, I.D., 0.69 mm) with an inner filament for easy dye filling.
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The dye should be dissolved in 0.2 M KCl and filtered (e.g. 0.2 μm spin filters for the microfuge). A 3% solution of rhodamine dextran (Molecular Probes) works quite well. Dye can be made in advance and stored at 4°C in a sealed tube. To fill pipette tips with dye, place a droplet of dye (about 0.5 µl) at the back of the pipette.
Labeling gastrula stage cells
Embryo preparation and mounting
Embryos should be removed from their chorions in agar-coated dishes using fine forceps. After removing the chorions, transfer the embryos with a fire-polished pipette. Experimental slides are made by painting several coats of nail polish in a ring of about 3 cm diameter, on a glass slide. Mount embryos in methyl cellulose (see METHYL CELLULOSE MOUNTING, page 4.7) to permit easy manipulation and reorientation. Chilled methyl cellulose is stiffer, but tends to get bubbly as it warms up, therefore it is best to use methyl cellulose at room temperature.
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Be sure to loosen the embryo from the methyl cellulose before trying to move it with a pipette.
Labeling cells
It is easiest to label cells if the embryo is oriented such that the surface to be labeled is at the top, lying flat. The ideal situation is to have the embryo oriented so that the cells to be labeled are easily visible in reference to landmarks such as the embryonic shield or the margin.
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Sometimes cells along the pipette track tend to be labeled by dye leaking from the tip. The tip may appear to have passed through these cells, but there is still continuity with their membranes. Single cell labels can be achieved by stopping dye injection as soon as one cell starts to label. Because of this problem, it is important to check the fluorescence without Nomarski optics (it will be much brighter) in order to see all of the cells that have been labeled.
Localizing the labeled cell
For mapping purposes, it is best to check the cell's location using three different orientations of the embryo. In the orientation in which the embryo was labeled, check the cell's distance from the margin under the 40x water immersion lens. Then turn the embryo onto its side and check the cell's radial depth at 40x. It is important to have a fairly precise side orientation, or else the cell may appear deeper or shallower than it is. The depth can also be assessed by focusing up and down in the original orientation, although this method gives no information about the relationship of the cell to the hypoblast. Finally, assess the position with respect to the dorsal midline by orienting the embryo animal pole up and looking at it under low power (10x objective).
Labeling single cells in older embryos
Mount embryos in agar as described in the section on AGAR MOUNTING (see page 4.5). Orient the embryo so that the cell of interest is visible with Nomarski optics and accessible with the pipette.
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Label the cell as described above, and quickly remove the pipette from the embryo by moving the embryo away with the stage controls.
Materials
Labeling rig:
• vibration-free table • oscilloscope
• micromanipulator • stimulator (optional)
• amplifier • bath ground
• probe • microscope with fluorescence light source
• electrode holder • 10x and 40x water immersion objectives
Pipettes:
• Sutter thick wall glass with capillary • electrode puller
• dye (fluorescent conjugated dextran) • electrode holder
• Hamilton syringe needles • 0.2 and 0.5 M KCl solutions
Miscellaneous:
• squirt bottle of Embryo Medium
• squirt bottle of distilled water for rinsing
Gastrulae mounting:
• Embryo Medium • 3% methyl cellulose (in Embryo Medium)
• agar-coated dishes • experimental slides
• fire polished pipettes • dissecting microscope
• hair loop • forceps
Older embryos:
• 1.2% agar in Ringer's • water bath at 42-44ºC
• experimental slides • fine probes