scientificprotocols authored about 8 years ago
Authors: Timothy Walston and Jeff Hardin
Adapted from Imaging in Developmental Biology (ed. Sharpe and Wong). CSHL Press, Cold Spring Harbor, NY, USA, 2011 (in press).
The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. The agar mount described in this protocol is an easy way to prepare live C. elegans embryos for microscopic visualization. The mount slightly embeds the embryo in agar to hold it in place. The mount also slightly compresses the embryo to provide consistent orientation such that every embryo will be positioned with either its right side or its left side facing the objective. Other techniques can result in random orientations that complicate analysis and make identification of individual blastomeres more challenging.
Protocols for collection and analysis of four-dimensional (4D) data can be found in Acquisition of 4D DIC Microscopic Data to Determine Cell Contacts in Caenorhabditis elegans Embryos (Walston and Hardin 2010a) and Analysis of 4D DIC Microscopic Data to Determine Cell Contacts in Caenorhabditis elegans Embryos (Walston and Hardin 2010b). An ablation technique for killing individual cells within the embryo (to facilitate the study of cell interactions within the C. elegans embryo) is described in Laser Killing of Blastomeres in Caenorhabditis elegans (Walston and Hardin 2010c). An alternative method for mounting embryos that involves raising a coverslip on grease “feet” is found in Suspended Embryo Mount for Imaging Caenorhabditis elegans (Mohler and Isaacson 2010).
The total time needed for the experienced is 10-15 min and for the novice is 30 min.
Figure 1. Isolation of C. elegans embryos and preparation for mounting on a slide. (A,B) Gravid hermaphrodites are cut in half with 27 × 1/2-in. needles. (C) At a higher magnification, embryos are sorted and are grouped using an eyelash. (D) Embryos and M9 buffer are transferred using a mouth pipette.
It is important to cut as close to the vulva as possible to release newly fertilized embryos in the uterus. This step can also be conducted by cutting the worm in half with a #15 curved blade scalpel.
3.Sort the embryos using the eyelash brush, and brush them together into a group of ~10 embryos (Fig. 1C).
Embryos will tend to stick slightly to each other when grouped. If you desire a certain stage of embryogenesis, it is at this point that the embryo stage should be assessed and the embryos sorted appropriately. Two-cell stage embryos are the easiest developmental stage to collect.
4.Make an agar pad:
Figure 2. Making an agar pad. (A) Three slides are placed on the bench, and the outer two are taped down to the bench. (B) A drop of molten 5% agar is placed onto the middle slide. A fourth slide is then placed perpendicular to the three original slides. The top slide is compressed over the taped slides. (C) The finished slide is sealed with valap. Using a toothpick to make hash marks in the valap (arrows) aids in finding the grouping of embryos on the compound scope.
5.Once the agar has set, use a razor blade to trim excess agar from the edges of the slides. Carefully slide apart the untaped slides, so that the agar pad is left in the center of one slide. See Troubleshooting.
6.Prepare a mouth pipette:
7.Using the mouth pipette, transfer the grouping of embryos (from Step 3) and ~20 μL of M9 buffer to the corner of the agar pad on the microscope slide (Fig. 1D).
8.Brush the embryos out of the M9 buffer into the center of the slide using the eyelash. Position the embryos in a single layer side by side.
9.Set the edge of a coverslip at the side of the agar pad opposite the M9 buffer and slowly drop it so that the coverslip lands on the embryos before it contacts the M9 buffer. Use a tissue to wick excess buffer from the edges of the coverslip, and wick air bubbles from under the coverslip. See Troubleshooting.
10.Trim excess agar from the edges of the coverslip using a razor blade. Seal the edges of the coverslip with melted valap for agar mounts using a paintbrush (Fig. 2C).
Mounting C. elegans embryos on agar mounts provides a stable long-term environment for microscopic analysis of development. The slight compression from the coverslip will result in embryos reproducibly positioned with either the left or the right side facing toward the objective lens. During later stages of embryogenesis, embryos turn such that left-side views become dorsal views and right-side views become ventral views. Embryos on agar mounts will survive and will hatch from the eggshell on the mount. Embryos prepared with an agar mount are amenable to both light microscopy (with differential interference contrast [DIC] optics) and confocal microscopy.
Alternative methods for mounting embryos, such as poly-L-lysine-coated slides with grease feet (see Suspended Embryo Mount for Imaging Caenorhabditis elegans [Mohler and Isaacson 2010]) or polymer beads, are often used to avoid compression to the embryo. However, these techniques typically result in random embryo orientation, which can complicate analysis of development. Additionally, the slight compression of the coverslip seems to have little to no effect on development; in most cases, avoidance of such compression is unnecessary.
In conclusion, preparing C. elegans embryos on an agar mount is a simple technique that can be easily mastered and is regularly performed by undergraduates in the investigators’ laboratories. It provides a consistent embryonic orientation and environment that is suitable for long-term microscopy of C. elegans embryos.