This protocol is significant, because it allows you to do immunohistochemistry on the notum of Drosophila pupa, even after they have been previously wounded, and live-imaged, without disrupting the architecture of the tissue. This technique allows you to take advantage, of the wide range of antibody stains already available, to stain cells, structures, and proteins, in the notum epithelium. We have used this technique for immunochemistry, and DNA-staining.
However, this dissection could be used as a starting point for other techniques, such as in situ hybridization. This protocol can be difficult, because pupa are small and delicate, so it requires a steady hand to perform the dissection. And it gets easier with experience, so practice on unimportant pupa before important ones.
Begin by carefully removing three to four pupae at stage P5, using a dissection scope, and collect them on the microscope slide next to the tape. Next, place the pupae at least one pupa width apart, onto the tape, with their ventral sides down. Now, place a drop of adhesive glue on a parrafin film, or in a centrifuge tube lid.
Next, dip the end of a 0.1 to 10 microliter pipette tip in the drop of adhesive glue, and tap the pipette tip twice on a cover slip, one centimeter by one centimeter away from a corner, creating a line of adhesive glue, approximately half the length of the pupa. Next, preset a P2 pipette at two microliters, and a P200 pipette at 200 microliters volume, and fit them with tips. Now, insert forceps near the side of the head, and gently remove as much of the pupil case as possible, working from the anterior to the posterior.
Next, grasp the pupa's developing legs, with a pair of blunt forceps, and carefully pull the pupa from its case. Lay the pupa in the corner of the cover slip. Grasp the pupa at the posterior abdomen, or the developing wing, with blunt forceps.
Lift and place the ventral side of the pupa down, into the line of adhesive glue. Quickly fill the P2 pipette, with two microliters of single-strength PBS, containing 0.1 millimolar calcium, and holding it in the air, expel just enough to form a small bubble at the tip. Touch the small bubble of the solution, to one side of the pupa at the base of the thorax, then repeat the procedure on the other side.
Next, fill the P200 pipette, with 200 microliters of single-strength PBS, with 0.1 millimolar calcium, then place the pipette's tip over the thorax, and expel the contents to submerge the pupae completely, and the remainder of the adhesive glue will solidify immediately. Now, remove approximately 100 microliters of the PBS solution, so the pupa is barely submerged, before proceeding immediately to the next step. To dissect the notum, grasp a pair of micro dissection scissors, bracing one side of the handle against the dominant hand's index finger and middle finger, so the thumb of the dominant hand applies the cutting force.
Next, stabilize the neck of the scissors against the middle finger of the non-dominant hand, while bracing the cover slip, with the ring finger of the non-dominant hand. Now, snip at the middle of the dorsal abdomen to create a small hole of approximately 0.2 to 0.5 millimeter. To isolate the dorsal tissue, make small 0.5 to 0.75 millimeter cuts through the integument, from the posterior to anterior, and in the end, these will encircle the dorsal tissue.
Further, repeat posterior to anterior cuts on the other side of the pupae. Rotate the dissection stage, to allow a clean cut through the head if necessary. Determine if the notum has been isolated, by seeing if it is easily moved.
Add approximately 200 microliters of single-strength PBS to the center of the cover slip, and make a channel connecting it to the original dissection droplet, by gently dragging the pipette tip across the cover glass, from the new droplet to the original one. Using a pair of blunt forceps, gently push or drag the isolated notum to the center of the cover slip and rotate, so the interior side faces upward. Hold the notum down with blunt forceps, by pressing into the abdominal or head section.
Using a pair of sharp forceps and gentle expulsions of single-strength PBS, from a 200 microliter pipette, remove any remaining fat body, muscle bands, or hemo lift, to expose the monolayer epithelium fully. Once the tissue is clean, use a 200 microliter pipette to remove as much of the PBS solution as possible, monitoring with the dissection scope, to avoid aspirating the notum. After maximum liquid removal, use an absorbent tissue, to carefully wipe away the rest of the adhesive glue, pupil carcass, and other debris on the cover slip.
Now, add 150 to 200 microliters of 4%PFA, and fix for 20 minutes at room temperature. And depending on the dissection speed, one to three more pupae can be dissected, during the first pupa's fixation on separate cover slips. Remove the PFA, and replace it with single-strength PBS, to wash the notum once for 30 seconds.
If proceeding with antibody staining, perform three five-minute washes, in single-strength PBS or PBST, to permeabilize the tissue if the antigen is intracellular. The sample can be stored in single-strength PBS, with 0.02%sodium azide overnight in a humidified chamber. Following staining, prepare a new cover slip referred to as a topper, with supports.
Create a gap of approximately 200 micrometers, by using spacers made from 22 by 22 cover slips, approximately one centimeter apart, in the middle of the topper. To adhere, paint the seam of the spacer furthest from the center, with a thin layer of nail polish, and then let it dry. Remove as much of the aqueous solution as possible from the sample, and immediately apply two drops of anti-fade mounting medium to the sample.
If necessary, use clean sharp forceps to position the notum in the anti-fade mounting medium droplet center. Place the cover slip with the notum, onto an approximately 10 by 40 millimeter foam support to elevate the sample so it does not adhere to the work surface. Under a dissection scope, slowly lower the topper onto the sample.
Once the anti-fade mounting medium meets the topper, gently release, and allow capillary action to pull the topper down. Place another piece of foam onto the topper, and use a standard microscope slide as a weight, to gently coax the anti-fade mounting medium between the sample cover slip, topper, and spacers. After 5-10 minutes, use an absorbent tissue to wick away any excess anti-fade mounting medium, by gently touching the edges of the cover slip.
Gently apply nail polish to each corner of the cover slips to adhere them together. To prevent the cover slip from shifting and damaging the dorsal tissue, avoid coating edges first, and once the nail polish in the corners dries, paint all edges of the cover slips to seal. This protocol allows imaging of the fixed samples, using antibodies and cellular stains.
This protocol can be used on samples that have been previously wounded and live-imaged. A comparison of live and fixed images, shows that the protocol preserves the tissue architecture and morphology. The protocol allows different views of a dissected notum.
The apical view is ideal for visualizing epithelial cell borders and nuclei, below the cuticle. The basal view better reveals basal structures at the wound margin. Be sure not to bend or warp the notum during dissection.
Cut a flat piece by avoiding the lateral edges. Don't cut too close to the ventral sides, otherwise it will deform during mounting. After dissecting a pupil notum, many other techniques could be applied, such as electron microscopy and cross-sectioning.
These techniques could allow previously imaged structures to be investigated in immense detail.
