Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The overall goal of this procedure is to dissect imaginal discs from drosophila larvae For immunohistochemistry, this is accomplished by first removing the head complex consisting of mouth, hooks, eye, and tenal discs, and brain from the larvae. The second step is to fix the head complex and incubate it with primary and secondary antibodies. Next, the eye and tenal discs are separated from the head complex.

The final step is to mount the imaginal discs in anti bleaching agent on a glass slide. Ultimately fluorescent or LAC Z reporters and antibody staining can reveal the spatial and temporal expression of genes and proteins within the imaginal tissues using fluorescence microscopy. Though this technique can be used to provide insight into gene and protein localization in the enten imaginal disc.

It can also be applied to other imaginal tissues, such as the wing leg, and halt.Here. To begin the experiment, fill a 35 millimeter Petri dish with dissection buffer. Place 10 to 20 larvae in the Petri dish and allow them to swim around for five minutes.

To self clean with a pest, your pipette create one large pool of dissection buffer in the middle of a silicon-based dissection plate. Place three smaller pools surrounding the large pool and use forceps to transfer the larvae to the bottom pool. Next, using number five forceps, transfer a single cleaned larvae from the small pool to the larger pool of dissection buffer.

To begin coarse dissection, use one pair of forceps to grab the mouth hooks and the other pair of forceps to grab the larvae gently. At one third its body length holding the mouth hook steady. Quickly pull the rest of the body away with a second pair of forceps.

Release the larva from the forceps and allow the guts to spill out, enabling the imaginal discs to remain in their normal confirmation. Then grasp the mouth hooks and use the other pair of forceps to remove the lower two thirds of the larvae, including the inner guts. Next, gently remove the salivary glands, leg discs, and other tissue.

The only tissues that should remain are a small amount of overlying cuticle. The mouth hooks, eye and tenal discs, brain hemispheres, and ventral ganglion. After a maximum of 20 minutes, transfer all dissected tissues to the paraform aldehyde lysine pariah.

Date fixative or PLP. Do not allow imaginal discs to remain in dissection buffer for longer than 20 minutes, or they will degrade following dissection. Transfer the dissected tissues in 100 microliter increments to a watch glass containing cold PLP.

Use a pair of forceps to ensure that the dissected tissues are completely submerged. Incubate dissected tissues in cold PLP fixative for 45 minutes at room temperature without agitation. Next, transfer 100 microliters of dissected tissues from PLP to wash buffer.

Ensure tissues are completely submerged and incubate them at room temperature without agitation for 45 minutes. Then transfer the dissected tissues to a 1.5 milliliter micro fuge tube. Remove the wash buffer and replace it with 100 microliters of blocking solution.

Incubate the tissues at room temperature with gentle rotation on a tabletop rotator for 10 minutes. After blocking, remove the solution and replace it with 100 microliters of primary antibody solution in 10%Normal goat serum. Incubate the tissues at room temperature with gentle rotation for 16 hours depending on the strength of the antibody.

Next, remove the primary antibody and replace it with 750 microliters of wash buffer. Place the tube on a mutator at room temperature for 10 minutes. Remove the tube from the mutator and allow the heads to settle to the bottom of the tube.

Then remove the wash buffer and add 100 microliters of secondary antibody solution in 10%Normal serum. Incubate the tube at room temperature with gentle rotation for at least two hours depending on the strength of the antibody. After incubation, remove the secondary antibody solution from the tissue.

Replace it with 500 microliters of wash buffer and allow the tissue to settle to the bottom of the tube. Place three pools of wash buffer on a silicon dissecting plate. Transfer all dissected tissues to the large pool of wash buffer that has been placed on the dissecting dish.

This helps to remove excess secondary antibodies to begin fine dissection, place the head complex ventral side down and clasp the cuticle by the mouth hooks. Place a second pair of forceps in the space between the brain and eye disks and swiftly pull the brain away from the mouth hooks to remove the two brain lobes while continuing to hold onto the mouth hooks. Pinch off the tissue as close as possible to the connection between the antenna section of the eye antenna disc and the mouth hook.

Continue until all desired tissue has been dissected. Next, add two small pieces of tissue paper the size of cover slips three inches apart on the dissecting dish. Place a glass slide onto these pieces of paper to prevent the slide from sticking to the silicon base of the dissecting dish.

Using a P 20 pipette men, add nine microliters of anti bleaching agent to the middle of the glass microscope slide to prevent fluorescent light bleaching of the tissue with the same uncut tip, gather all I and tenal discs and add them to the drop of anti bleaching reagent. Next, use forceps to separate and spread out the eye and tenal discs within the drop of anti bleaching reagent until no imaginal discs are touching. Using a fine paint brush gently lower a cover slip onto the drop of anti bleaching agent to prevent the formation of air bubbles.

Finally, store slides at negative 20 degrees Celsius until ready to view and image the ion tenal discs using light or fluorescence microscopy. This method reliably produces high quality material for analysis as conveyed by these confocal images of imaginal discs. The discs were treated with a foid and conjugated fluoro four, which binds to f actin and therefore outlines each cell.

One of the most striking features of the ental disc is the morphogenetic furrow, which can be seen as an indentation within tissue running along the dorsal ventral axis. As the furrow progresses across the eye field, the sea of disordered cells is transformed into an ordered array of periodically spaced unit eyes or Oma Tedia. Verification of clonal expression and analysis of the I imaginal disc are possible downstream applications of this method.

The top three panels display discs that have been stained with different antibodies such as ilis, A DPP, LAC Z transcriptional reporter, and cell specific markers such as embryonic, lethal, abnormal vision, or vy, which encodes a pan neuronal, RNA binding protein. The lower three panels show discs in which GFP is used to mark populations of cells. Once mastered the course, isolation of head complexes from 10 to 20 larvae will take approximately 10 minutes, and additionally, the fine isolation of the ion tenal disc from these complexes will take about 10 minutes as well.