The overall goal of this procedure is to prepare adult drosophila eyes for thin sectioning, allowing for the detailed assessment of mutant eye phenotypes, such as planar cell polarity defects. This is accomplished by first cutting off the heads of anesthetized flies. The eye tissue is then fixed and embedded in an epoxy resin.
Once the resin is solidified, the embedded eyes are trimmed. For sectioning, the final step is to thinly section the eyes using a microtome equipped with a diamond knife. Ultimately, analysis of the eye section will allow the characterization of eye phenotypes of novel mutants or the quantification of genetic interactions between genes of a signaling pathway, such as the PCP pathway.
Both will help to characterize the physiological role of newly identified genes. This method can help answer key questions in the drosophila field, such as how novel mutations affect eye development, or how genes interplay in genetic interaction assays. Several steps of the procedure of eye embedding, such as aligning the eyes in sectioning molds are way easier to understand, which really rather than in text form To begin put on a pair of gloves in preparation for the fixation of the fly heads and glutaraldehyde and osmium, both of which are toxic chemicals.
Next anesthetize flies using CO2 and tap them out onto a fly pad protected by watman paper under a dissecting microscope, typically sort out six flies plus an additional fly to ensure six embedded fly heads per genotype hold and gently press the fly thorax with tweezers and then remove the fly's head using a scalpel, stabilize the fly head by touching the neck with the tweezers and carefully cut off a small part of one eye in order to enhance penetration of the fixative. Touch the head with tweezers or the scalpel on the surface of the cutoff eye to transfer the head into glutaraldehyde phosphate fixative on ice. Be sure to wear gloves throughout the fixation process.
First label all molds in order to keep track of different genotypes. Then fill each mold with 100%resin taking care not to overfill them. The top should be flat across the surface.
Using a transfer pipette, remove the 50%resin from the heads. Replace with 100%resin and incubate for three to four hours at room temperature. As the heads equilibrate in the resin over the course of the incubation, they will sink to the bottom of the tube.
Hit a toothpick on a hard surface to create a little hook. Using this tool, transfer a single fly head into each mold. Place aluminum foil on the working area of the dissecting microscope to protect it against spilled resin.
With the aid of the dissecting scope, use the dissecting needle to swirl the head slightly and carefully. Move it to the bottom of the mold, close to the pointed end. Carefully align the surface of the intact eye with the front wall of the mold with a neck pointing down.
Be sure to keep the tangential surface of the eye, roughly half a head diameter away from the mold wall. If the eyes move when removing the dissecting needle, reposition the eyes desired, quickly retract the needle slightly away from the head, and then slowly remove the needle completely. Once all the heads have been aligned, recheck all eyes to make sure they did not move.
When you removed the needle out of the resin, if any or askew realign the head, transfer the molds to an oven and bake it 70 degrees overnight. After baking overnight, remove the hardened blocks from the molds by bending the molds. Keep track of which block corresponds to which genotype by keeping them in labeled containers.
To trim the blocks, mount a block onto a microtome chuck, such that the eye is facing upwards under the dissection scope. Using a Teflon coated razor blade, carefully cut off only the top layer of the block. This leaves a clear surface through which the eye can be seen, and the plane of sectioning can be established.
Next, cut off excess plastic on both sides of the head and on the front until most of the plastic around the head is cut off. It is best to slowly approach the head by cutting off multiple thin layers of plastic using a clean razor blade. Carefully remove thin layers from the top of the eye in the exact desired plane of sectioning.
Be sure to use fresh areas of the razor blade for each cut and continue just until the outside surface of the eye is reached. The resulting trimmed block will be a three-sided pyramid with a slightly tilted cutoff top that corresponds to the plane of sectioning. Mount the block in the microtome and begin sectioning.
Until 20 to 30 sections have been generated, the resulting sections will float on the surface of the water in the reservoir. Place a slide on a heating plate set to 100 degrees Celsius. Then lift the first batch of sections with a flattened end of a wooden Q-tip from under the water surface using a rotating movement, and then transfer them into a drop of water.
On the slide, wait until the water has evaporated and the sections are stuck to the slide, and then repeat with another 20 to 30 sections. Typically, sections of three eyes positioned in six water drops fit well on one slide. Once all the sections are collected on the slide, they can be stained and mounted for microscopy In a tangential section through the eye of a wild eye fly.
The TIA display normal polarity and are well oriented with respect to the dorsal ventral line of symmetry, which is illustrated by a yellow line in the schematic below. In contrast to the well oriented tia of the wild type eye here in a strabismus mutant eye, the tial polarity is lost, and even though the full photoreceptor complement is present, rotation and ality are randomized. Also, note the absence of the pigment of the pigment cell lattice and the individual photoreceptor cells.
Since the strabismus mutant was in a white minus genetic background, drosophila ro kinase or drop is a homozygous lethal mutation, which thus requires clonal analysis. Here, Dr.Homozygous mutant clones are marked by the absence of pigment and show rotation defects, as well as structural defects, including missing or excess numbers of photoreceptor cells on the single cell level. Examples of mutant and wild type photoreceptors are indicated by open or filled blue arrowheads respectively.
After watching this video, you should have a good understanding of how to embed and section fly eyes. Within about three days. You should routinely be able to analyze eye phenotypes in detail, allowing you to address the function of novel genes during eye development.
