The overall goal of this procedure is to prepare the abdominal epithelium of drosophila puy for immunohistochemistry. This is accomplished by first bilaterally, dissecting stage drosophila puy, and removing the internal organs such that only the abdominal epithelium composed of proliferating imaginal. His osteoblast and larval epidermal cells remain attached to the pupil membrane.
The tissue is then fixed incubated with antibodies removed from the exterior pupil case and visualized through immunofluorescence microscopy to show the gross morphology of the abdominal epithelium and expression of proteins of interest. Hi, I'm John Yoder. My lab is at the University of Alabama in the Department of Biological Sciences.
Hi, I'm we won. I'm a peer student Yoda lab. Today we're gonna show you our method for dissection of the drosophila, cupe, and immunohistochemistry of the drosophila pupa abdomen.
This method can be used to investigate processes of epithelial morphogenesis and tissue reorganization. We specifically use this technique to investigate the development of sexually dimorphic morphology of the adult drosophila abdomen From a 25 degrees Celsius population vial without adults identify. Recently immobilized puy puy with cuticles that haven't yet begun to tan are considered zero hours a PF.Using a wetted paintbrush, gently remove these puy and place them on the lid of a humidified chamber Using the paintbrush and PBS gently wash away debris from the puer cases if necessary.
Sort the puy by sex at zero hours. A PF male goad primordial are easily seen through the pupil cuticle. Approximately two thirds of the way down the body, the female goad primordial are smaller and not as easily identified.
Now, place the pube on the wetted filter paper in the humidified chamber. Replace the lid and return them to a constant temperature environment. When the animals have reached the developmental time point of interest, proceed with the dissection.
Be aware that puby processed 24 to 32 hours A PF are most prone to epithelial cell loss during subsequent processing. Prepare for the dissection by endearing a piece of double-sided tape along one side of a transparent slide. Then use forceps to gently grasp each pupil between the anterior sphericals and place them on the tape dorsal side up.
Line up a maximum of 10 PPE in this manner or risk working too slowly. In five to 10 minutes, the PPE will be completely endeared, but before this happens, use a paintbrush to space them out evenly and position them in parallel. Prolonged drying at this stage will diminish their quality.
So move quickly. Now use a surgical scalpel to dissect each pupil A bilaterally. Make a single rapid cut from the anterior to the posterior end of the pupa.
If done properly, the cut will bisect both anterior and posterior spherical pairs producing symmetric left and right halves to loosen the pube from the tape. Use a paintbrush to apply a small amount of PBS to each dissected pupa using surgical forceps. Grasp one pupa half by its anterior end and immerse it in a PBS filled rinsing dish on the dissecting microscope stage while still holding the pupa, use a pipetter to gently wash away the internal tissue by pumping PBS into the abdominal cavity.
Avoid applying too much pressure or cells will be lost from the epithelium. Immediately submerge the clean tissue in 400 microliters of room temperature fixation buffer, clean all of the pupa halves. Similarly with practice dissecting and washing 20 PU halves should take less than five minutes.
Allow the puy to incubate in the fixation buffer at room temperature for an hour, and then rinse them three times in 400 microliters of PBS for five minutes per wash. To store the samples first, rinse them twice in 100%methanol, then transfer them to 100%ethanol for storage. These samples can be stored in 100%ethanol at minus 20 degrees Celsius for up to three months without any loss of cells or epitope reactivity.
To process stored samples, they must first be rerated in PBS through an ethanol series. Proceed with the immunohistochemistry by blocking the samples in PBS with 2%BSA for an hour at room temperature and without rocking. Rocking samples, greatly increases cell loss from the abdominal epithelium.
Then replace the blocking solution with the primary antibody diluted to the appropriate concentration in PBS. Incubate the samples overnight at four degrees Celsius without rocking and in a closed humidified chamber to prevent evaporation the next day. Wash the samples three times in PBS without rocking and for 10 minutes per wash.
Then block the samples for an hour without rocking in PBS with 2%BSA. Replace the block solution with room temperature secondary antibody in PBS. Then incubate the samples in the dark for three hours and without rocking.
Follow the secondary antibody with three wass in PBS for 10 minutes each if appropriate. Counterstain the puy, for example, nuclei can be stained with a 10 minute incubation in DPI diluted by PBS. Next, equate the samples in mounting media for a minimum of 30 minutes.
To mount the samples, grasp each pupil case anteriorly, avoiding the internal pupil membrane with a second pair of forceps. Gently grasp the internal pupil membrane by the head. Then gently pull the sample from the pupil case.
Transfer the unca sample to a depression slide with 75 to 100 microliters of mouthing media. Several samples can be mounted on a single slide. Now use a probe and forceps to position the samples uniformly in the depression.
Well rotate them so that their lateral surfaces face upward and remove any air bubbles with a probe. Finally, gently lower the cover slip onto the samples without introducing air bubbles for this preparation. Structured illumination microscopy will be comparable to confocal techniques.
This is a projection of 50 confocal images with 2.5 microns between slices. The tissue is at 26 hours a PF, and the staining shows the expression pattern of wingless protein and grail driven GFP and the nuclei. The anterior is to the left and dorsal features are to the top of the screen.
This movie demonstrates how the gross morphology of samples is preserved by the procedure. Thus allowing three dimensional reconstruction of image stacks. West mastered several dozen pupil may be persisted and placed in fixation buffer in less than 30 minutes.
Additionally, samples prepared by using this protocol are amenable to institute hybridization making this a valuable protocol for gene expression studies.
