实验概要

The Drosophila larval brain is a well-established model for investigating the role of stem cells in development. Neuroblasts (neural stem cells) must be competent to generate many thousands of differentiated neurons through asymmetric divisions during normal development. Studies in fly neuroblasts have been instrumental in identifying how the establishment and maintenance of cell polarity influence cell fate, and they have produced a wide array of molecular cell-polarity markers. Moreover, neuroblasts and their progeny can be positively identified using a variety of cell-fate markers. This article describes procedures for the collection and processing of Drosophila larval brains for examination by immunolocalization of cell-fate and cell-polarity markers. The protocol can be used for dissecting, fixing, and staining brains from larvae at any developmental stage. The number of brains processed using this method is limited only by how many brains can be dissected in 20 min, which is the maximum amount of time dissected tissues should remain in buffer before fixation. This protocol can be used for simultaneous costaining of multiple proteins.

实验步骤

Dissection of Larvae

• 1. Fill the wells of dissection dishes with 200-400 μL of cold Schneider’s insect medium.

• 2. Prepare to dissect the larvae by rolling them onto their dorsal side so that the denticle belts are facing upward.

• 3. Using a pair of forceps, gently grasp the larva just posterior of the midpoint. With a second pair of forceps, grasp the anterior end of the larva at the base of the mouth hooks.

• 4. Carefully tear the cuticle behind the mouth hooks using a side-to-side motion while slowly drawing the mouthparts out away from the body. The brain will remain attached to the head and be clearly visible among the gut and salivary glands. Remove any excess tissue, but leave the brain attached to the mouth hooks.

  Leaving the brains connected to the mouth hooks will help the brains sink to the bottom of the tube during washing steps below. Moreover, the mouth hooks are dark in color, which makes it easier to see the brains during experimental manipulations.

• 5. After dissection, place the brains in a 0.5-mL microcentrifuge tube containing cold Schneider’s insect medium.

  Do not let the tissue sit in Schneider’s insect medium for >20 min.

Fixation and Staining

• 6. Remove the Schneider’s insect medium from the samples.

• 7. Add 500 μL of fix solution and incubate with rocking for 23 min at room temperature.

• 8. Quickly wash the brains twice in ~500 μL of PBSTX at room temperature. Wash again in PBSTX twice for 20 min each at room temperature.

  Once fixed, samples can be held in extended washes to synchronize them before proceeding with further processing.

• 9. Incubate the samples in ~500 μL of block solution (D) for at least 30 min at room temperature.

• 10. Incubate in primary antibody diluted in PBSBTX for 4 h at room temperature or overnight at 4°C.

  Conditions are dependent on the specific antibody being used. For example, Dpn staining is better when incubated for 3-4 h at room temperature rather than overnight at 4°C.

• 11. Quickly wash the brains twice in PBSBTX at room temperature. Wash again in PBSBTX twice for 30 min each at room temperature.

• 12. Incubate the samples in secondary antibody for 1.5 h at room temperature or overnight at 4°C. Protect the samples from light after this point.

  Secondary antibodies are typically diluted in PBSBTX.

• 13. Quickly wash the brains twice in PBSTX at room temperature. Wash again in PBSTX twice for 30 min each at room temperature.

• 14. Equilibrate the brains in ProLong Gold at room temperature.

  Samples can be stored in the dark at room temperature.

Mounting Samples

• 15. Adhere two 22- × 22-mm coverslips to a microscope slide using a small amount of 70% glycerol, leaving an ~5-mm space between them.

  These coverslips act as spacers to prevent the brains from being deformed by the 24- × 40-mm coverslip in Step 19.

• 16. Transfer the brains to the slide using a pipette with the tip cut off.

• 17. Using forceps, remove all excess tissue including the optic discs from each brain. Be sure to leave the ventral nerve cord intact, because it will aid in proper orientation of the brain on the slide.

  See Troubleshooting.

• 18. Orient the brains ventral side down.

  If the ventral cord is intact, the brain will sit in the appropriate upright position. Without the ventral cord, it is difficult to keep the brain in the proper position, and it will tend to end up resting on its anterior or posterior surface.

• 19. Place a 24- × 40-mm coverslip over the samples and backfill the space between the slide and the coverslip by pipetting a small amount of mounting medium along the edge of the coverslip.