Most of the images in this primer were collected on a Pixera digital camera mounted on a Zeiss Axioplan microscope (DIC images are 63X oil immersion or 40X multi-immersion (with water)). Brightfield whole embryo images were made using the 10X dry lense. The images were collected at the maximum possible resolution using the Pixera image-processing program,and then saved as Adobe PhotoShop 3.0.5 files (PICT format). These in turn were further processed for color balance, cropped, and labeled. These files are about 500 pixels/hemisegment, so that a two-hemisegment image is about 1000 pixels wide. For installation on the Web pages, these were reduced to a maximum of 600 pixels in total width (maximum width per segment=320 pixels) and saved as JPEG files. This produces a dramatic compression of the image (the Photoshop files are several Mb, while the JPEG versions are 50-100 K), without affecting image quality very much. If the JPEG and Photoshop images are blown up several times, one sees some differences between them (JPEG is more pixilated and has less subtle color gradations), but at normal magnification they are almost indistinguishable. The Pixera digital camera files have less resolution than a Photoshop image generated from a scanned negative. Nevertheless, their resolution is still in excess of what can be displayed by a Web page. For publication, we still use scanned negatives. For documentation, storage, production of slides, etc., however, the Pixera files are excellent, and a great deal of time and energy is saved relative to trying to keep track of stacks of prints or slides.
The following is one dissection protocol. Each person will want to design a protocol that works well for them. What is described here may not be the way you eventually choose to do dissections, but at least it provides a guideline.
Clear embryos in 90% glycerol after staining. For dissection pick late 16 or early 17 embryos under the dissecting scope. These should have contracted nerve cords and fully segmented guts. Then put a single embryo on an uncoated glass slide with a few microliters of glycerol (enough to just spread out into a thin layer under an 18 x 18 mm coverslip). The amount of glycerol used under the coverslip strongly affects the muscle morphology. If only a small amount is used the preparation will often be quite flat; this will allow visualization & photography of a whole nerve branch in a single focal plane, but usually the muscles will not be easily distinguishable with DIC optics. If a larger amount is used the muscle morphology will be better, but the embryo will be more three-dimensional, so that nerves will move in and out of a given focal plane. You should experiment with this to define the optimum amount of glycerol.
For dissections we use tungsten needles mounted by threading tungsten wire (Ernest Fullam Corp.) into a 21 gauge needle in a dispo 1 ml syringe. Sharpen by electrolysis in a 1M KOH bath. Glass needles (made in a needle-puller) can also be used. Move the embryo a short distance out of the glycerol drop, so it is free of most glycerol but has a little bit associated with it. Roll the embryo so it is dorsal side up. Orient the embryo vertically in the field (head down) and use 2 needles to split it down the dorsal midline. You do this by making a small cut just posterior to the brain, then cutting down the dorsal midline toward the posterior, spreading the sides of the embryo out with both needles at intervals. When you reach the posterior end, cut directly toward the posterior and try to remove the proctodeum and hindgut if possible; in any case make sure the body walls are not connected to each other. Then you may want to cut off the head end, using the needles like scissors. Then cut toward the anterior along both sides of the brain, and fold the brain back toward the anterior. This will mess up the anterior (thoracic) region, but you're not looking at those segments anyway.
The rest of the dissection, which is very delicate, you may want to do with only one needle, and steady one hand with the other. Now you spread the sides of the embryo out and remove the gut. You can poke the gut initially and then push it down lightly against one body wall, then pull the gut free after the body wall is stuck. When both body walls are stretched out & the gut has been removed (pick off any loose yolk bits), gently drop a cover slip onto the embryo from one edge, letting it fall onto the pool of glyerol before it falls onto the embryo. This will spread the glycerol pool over so it covers the embryo. If it doesn't cover it (not enough glycerol or embryo too far from the pool), you can add more glycerol to the edge and set the slide vertically, so it can run under the coverslip. Now seal the edges with nail polish and examine under high power (40X multi-immersion lens with water or 63X with oil). This is for a Zeiss Axioplan. If using the 40X multi-immersion lense use the .3-.4 DIC setting even though the numerical aperture on this lense would suggest you should use the .5-1.3 setting.