Presentation of Embryos for Fluorescence Microscopy
You might think that the seemingly prosaic subject of mounting embryos on slides is not worth much discussion. But with several expression patterns to visualize in single specimens, and the ability to image them at cellular resolution in three dimensions with fluorescence microscopy, the more information each embryo can offer, and thus the more its presentation to the microscope matters. The main problem in mounting these embryos in the glycerol-based media that is widely used for fluorescence microscopy, is their softness caused by the long hybridization. If you're used to doing fluorescent antibody staining, you'll notice the difference: embryos incubated overnight in PBT for antibody staining are hard as rock compared to hybridized embryos. And since mounting alkaline phosphatase stained embryos normally involves ethanol dehydration (which seems to shrink and firm them) , and a mounting media that hardens (such as Permount), their fragility is not an issue. So to achieve the best presentation of these fluorescent in situ embryos, the challenge is to achieve the best balance between several physical forces. Two forces tend to spread the embryos out under the coverslip and flatten them: the coverslip has some small mass and the mounting media 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 media. Progressively greater downward force will result first in flattening, then warping, then rupturing your embryos; this is caused by too few embryos or too little mounting media. The counterforce of more embryos under the coverslip will remedy the problem, or using more mounting media. On the other hand, insufficient downward force will result in tilted or floating embryos and embryos stacked up on top of each other. This doesn't hurt the embryos themselves, but if you are using a confocal with an oil immersion objective, the up and down movement of the stage as you explore different optical sections will result in the slight shifting of the unstabilized embryo, and the images in your z-series won't be in register. In this case, reduce the amount of mounting media or number of embryos, or use a larger coverslip. This will also reduce the problem of excessive crowding of embryos on the slide, which can spoil the view of perfect specimens. Many people use a system of coverslip supports to avoid the problem of damaging fragile embryos, so do that if it suits you. Also, there are some glycerol-based media which eventually harden, so perhaps that's the best solution, but I haven't tried any yet.
Here's my current method for mounting these fluorescent in situ embryos, optimized for ~40 µl settled embryos in PBT and standard 22 mm x 40 mm, #1 thickness coverslips:
- After the final PBT washes, Rock 1x, 70% glycerol / 30% PBT, 10 minutes
- Let embryos settle, which may take a while because of the thick glycerol
- Drain the 70% glycerol as completely as you can
- Using a p200 micropipette cut with a razor at about the 10 µl level, pipette ~60 µl of your glycerol-based mounting media down onto the embryos, but don't fully empty the pipette. The embryos start to float up, and right away start to gently pipette up and down until you see that the embryos are thoroughly mixed in with the mounting media, usually at least 10 up and down cycles. To avoid mixing in air bubbles with the media, don't ever completely empty the cut micropipette.
- Pipette the embryos onto a microscope slide and lay down the coverslip and let the media spread to its edges. The embryos will take at least a couple of hours to fully equilibrate with the mounting media, but you can look at them right away. After some time, you can wick away some amount of liquid from the edges of the coverslip with the edge of a kimwipe, if you want to reduce the volume under the coverslip in order to get the embryos to lie in a flatter, more stable orientation. If you wick away too much, you will start to crush and warp the embryos, as discussed above.
Use your favorite anti-fade mounting media for these fluorescent samples. If it is glycerol based, you can store your slides at -20° C. for months with only slight diminuition of signal. I think that 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 I'm using cheap home-made stuff that is working perfectly well for most imaging situations, described in Reagents and Recipes.
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