Dissection and Fixation of Whole Mount Brains

Good tools are most important. Newly bought forceps are usually not fine enough for good preparations. They need to be sharpened with the help of sandpaper under a dissecting microscope (Fig. 1A). The sharpened forceps should then be used only for the dissections (use other forceps to grasp flies, etc.). It is also necessary to resharpen the forceps from time to time after regular use.

Dissection is preferentially performed in a black block dish filled with PBS. The black background helps to distinguish the opaque white brain tissue from other structures during preparation. If no black-block dish is available, put a piece of black paper underneath a conventional dish. For illumination use a cold light source. Place sheets of tissue paper on both sides of the block dish to deposit pieces of cuticle, fat body, air sacs, tracheae, and the like during dissection, and to clean the forceps from debris once in a while.

3.1.1. Dissection of Adult Brains

1. Grab the fly with one forceps at the thorax and with another at the base of the proboscis.

2. Remove the head from the body by pulling slowly. Then remove the proboscis. Alternatively, remove the proboscis and then grab the head from below by inserting one forceps into the foramen of the mouth parts and remove the head by pulling.

3. Open up the head capsule from ventral to dorsal (see Note 4) by "cutting up" (i.e., carefully breaking the cuticle with the two forceps; Fig. 1B) the region between antennae, forehead, and the top of the head.

4. Gently pull apart the right and left halves of the head capsule to isolate the brain. It is important to pull extremely slowly to minimize mechanical stress; otherwise the optic lobes will separate from the central brain. Alternatively, make two further cuts through the cuticle of the optic lobes (Fig. 1C,D) and then remove the brain piece by piece; this is safer for the beginner.

5. Once the brain is isolated, remove the esophagus, air sacs (Fig. 1E,F), trachea, and fat body from the isolated brain (see Note 5).

3.1.2. Dissection of Larval Central Nervous System

1. Grab the larva with one pair of forceps at the mouth hooks and with another pair at its anterior-middle part (about one-fourth of the way from the head) and pull

Fig. 1. Dissection of Drosophila brain. (A) Sharpening of a Dumont no. 5 forceps. (B-D) Opening the head capsule, main steps (see Subheading 3.1.1.; steps 3 and 4 and Note 4). (E) Isolated brain with air sacs (arrows), posterior view. (F) Brain with partly removed air sacs, anterior view. (G) Dissected brain with air sacs completely removed; only some tracheae are still present (arrowheads).

Fig. 1. Dissection of Drosophila brain. (A) Sharpening of a Dumont no. 5 forceps. (B-D) Opening the head capsule, main steps (see Subheading 3.1.1.; steps 3 and 4 and Note 4). (E) Isolated brain with air sacs (arrows), posterior view. (F) Brain with partly removed air sacs, anterior view. (G) Dissected brain with air sacs completely removed; only some tracheae are still present (arrowheads).

slowly. Usually, the larva breaks in the middle and internal organs spill out, whereby the central nervous system (CNS) remains attached to the mouth hooks.

2. Locate the CNS and remove other tissue (see Note 6).

3.1.3. Fixation of Whole Mounts

1. Using a glass Pasteur pipet (see Note 7) transfer the dissected brain together with a small volume of PBS to a second block dish filled with PFA (or other fixative; see Note 8). Keep dissecting for up to 20 min, pooling the brains in the second dish (5 to 20 brains, depending on your dissection speed).

2. Remove the diluted fixative and replace it with fresh fixative. Fix for another 40 min (see Notes 9 and 10).

3. Remove the fixative and wash the brains three times for 10 min in PBS and then 2 times for 10 min in PBT. Always remove the old buffer with a Pasteur pipet and immediately replace it with a fresh one; do not let the specimens dry in between (see Note 11).

4. Proceed with the immunostaining.

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