Presentation of Embryos for Fluorescence Microscopy

Having visualized several expression patterns, and with the ability to image them at cellular resolution in three dimensions using fluorescence microscopy, each embryo presents a wealth of information, and thus it is important that it is mounted properly for optimal image acquisition. The main problem in mounting embryos (in the glycerol-based media that are widely used for fluorescence microscopy) is that the embryos become quite soft and fragile following the long hybridization protocol. The difference between embryos processed for multiplex FISH and those for fluorescent antibody staining is striking: embryos incubated overnight in PBT for antibody staining are very rigid compared to hybridized embryos. The challenge in achieving the best presentation of these fluorescent in situ embryos is to balance the several physical forces acting on them on a microscope slide. Two forces tend to spread the embryos out under the coverslip and flatten them: the coverslip has some small mass and the mounting medium has surface tension which pulls the coverslip down as it spreads all the way to the edge. The two opposing forces are the resilience or 'springiness' of the embryos themselves and the volume of mounting medium. Progressively greater downward force will result first in flattening, then warping, and finally rupturing the embryos. Such problems can result from loading too few embryos or too little mounting medium on a slide. The counterforce of more embryos under the coverslip will remedy the problem, or using more mounting medium. On the other hand, insufficient downward force will result in tilted or floating embryos and embryos stacked up on top of each other. This does not damage the embryos, but when trying to image them by confocal microscopy using an oil immersion objective, the movement of the stage during optical sectioning will result in the slight displacement of an unstabilized embryo, which will shift the images in a z-series out of register. In this case, one can reduce the amount of mounting medium or the volume of embryos, or use a larger coverslip. This will also reduce the problem of excessive crowding of embryos on the slide, which often ruins the presentation of ideal specimens. Other means of preventing damage to fragile embryos are to use a system of coverslip supports or a mounting medium that eventually hardens.

The following method for mounting fluorescently labeled in situ embryos is optimized for ~40 µl settled embryos in PBT and standard 22 mm x 40 mm, #1 thickness coverslips.

  1. After the final PBT washes, Rock 1x, 70% glycerol / 30% PBT, 10 minutes.
  2. Let the embryos settle, which may take some time because of the viscosity of the solution. At this point, the embryos can be stored for months at -20° C. before mounting.
  3. Drain the 70% glycerol as completely as possible.
  4. Cut the end off a p200 micropipette tip (at about the 10 µl level) with a razor blade. Using this cut tip, pipette ~60 µl of a glycerol-based mounting medium onto the embryos without fully emptying the pipette. As the embryos begin to float up, start gently pipetting up and down until the embryos become thoroughly mixed with the mounting medium (this usually requires at least 10 up and down cycles). To avoid mixing air bubbles into the medium, do not completely empty the pipette during mixing.
  5. Pipette the embryos onto a microscope slide and apply the coverslip, letting the medium spread to the edges. The embryos will take at least two hours to fully equilibrate with the mounting medium, but they can be examined immediately, if desired. To reduce the volume under the coverslip in order to get the embryos to lie in a flatter, more stable orientation, one can wick away excess medium from the edges of the coverslip with a kimwipe. However, if too much liquid is removed, the embryos will start to be crushed, as discussed above.

It is suggested to use an anti-fade mounting medium for these fluorescent samples. If it is glycerol based, the slides can be stored at -20° C. for months with only slight diminution of signal. Specimens mounted in glycerol actually improve their presentation after some storage time, as the tissue continues to clear and the indices of refraction throughout the mountant even out. There are many commercially available media, but several inexpensive glycerol solutions, described in Reagents and Recipes, perform well for most experiments.

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