Um método rápido e eficiente para dissecar asas Pupal de Drosophila apropriado para Immunodetections ou ensaios PCR

The overall goal of this procedure is a rapid and efficient method to dissect pupal wings of Drosophila suitable for immunodetections or PCR assays. The procedure described here was developed by the demand to find a fast and efficient method to obtain pupal wing samples suitable to perform PCR assays or immunodetection protocols. Using a wet paintbrush, gently remove zero-hour APF pupa from culture vials and place them in new vials to complete the development period.

For example, if using a pupa that is 24 hours APF, it would left in the vial only 22 hours. At 22 hours, transfer the pupa to the microscope slide with the double-sided tape adhered to it. This transference could be done using a paintbrush to avoid damaging the pupa.

The pupa should be positioned forming a row with dorsal sides up and cephalic ends facing the same direction. After this, return the microscope slide to a constant temperature and allow the pupa to complete the appropriate developmental time. In this case, it would be 24 hours.

The microscope slide with the double-sided tape will be a transitory dissection platform to remove the pupal case. During the two hours on the microscope slide, the pupal case will dry, making it easily breakable with forceps. The first step in dissection is to remove the pupa from the carcass.

Then the pupa will be transferred to a different slide with double-sided tape adhered to it. The carcass will be left behind. This pupa will be stuck with the ventral side facing upwards, placed in the middle of the tape.

Before performing the wing cut, the pupa must be covered with a solution according to the procedure using immunohistochemistry. This solution will be affixative and the wing will be freed from the cuticle. Using forceps, gently remove the operculum from the pupal case.

Break the case with forceps, making a line along the side of the pupa to the caudal end of it. With the appropriate illumination, it is possible to visualize the internal shape of the pupa, making an evident space just above the hinge of the pupal wing. This space serves as a guide to perform the opening without damaging the pupa.

As the case is opened laterally, it will be possible to remove portions of it, picking them up by the open border and carrying them to the opposite side where the double-side tape will hold them. At the end of the dissection, the pupa will be almost naked. Only a piece of case will be covering the posterior tip.

With the aid of the forceps, gently push down the remainder of the case. This move will raise the interior end of the pupa, allowing both the head and thorax to stay in a preferential position to be stuck to the subsequent step. Take a new slide with double-sided tape and invert it on the pupa.

Then press slightly to make the pupa stick to the tape and gently slip the slide in order to remove the pupa from the rest of the cases. Invert the microscope slide. The pupa will be stuck on the dorsal side at the level of their head or thorax area.

For immunohistochemistry use, cover the entire naked pupa with 4%paraformaldehyde. After 10 minutes in affixative, with the aid of scissors, make a little incision at the hinge region of the wing or near it. Allow the affixative to reach the wing epithelium.

Then after five minutes, tear with forceps from the cuticle borders, expanding the hole and enabling the release of the wing epithelium from the cuticle sac. Once the wing tissue is released, leave it in the paraformaldehyde solution until completing 30 minutes of fixation. The method of dissection described in this video can be used to prepare high-quality samples of Drosophila pupal wings from different development stages for many types of techniques.

The protocol could be performed to dissect one pupa or several pupae at the same time. This helps to accelerate the procedure, making it easier to reach a large number of wings dissected. To remove the naked pupa from the opened case, it is important that the posterior tip will be small enough.

Although it is possible to remove a row of four or five naked pupa at the same time, it is not a relevant point, being able to remove those remaining attached to their case in successive steps using a single double-sided tape. This is confocal image of a 24-to 30-hour APF wing. The staining shows the expression of patched protein, the canonical receptor of hedgehog, the nuclei and actin filaments.

A similar dissection process could be used to obtain larger wing samples to isolate mRNA. This mRNA can be used to synthesize cDNA which can be used in PCR reactions. Here we show the products obtained to detect the expression of patched and actin.

The protocol offers a straightforward method for obtaining pupal wing samples that retain the familiar morphology of the wing. From these preparations, it is possible to obtain stacks of images that could be projected as 2D or 3D images to investigate distribution and/or the enrichment of proteins during the differentiation of the wing. A similar protocol can be used to collect a large pupal wing sample necessary to synthesize cDNA, a molecule essential in PCR reactions.

This procedure provides an alternative and complementary approach to the already-published methods for pupal wing dissection.