INTRODUCTION

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-polaritymarkers. 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.

RELATED INFORMATION

Immunofluorescent staining of cell-fate markers in larval brainneuroblasts is shown in Figure 1 and immunofluorescent staining of cell-polarity markers is shown in Figure 2 . When examining mutants that affect the development of Drosophila larval brains, it may be useful to employ other labeling methods, such as those described in EdU (5-Ethynyl-2''-Deoxyuridine) Labeling of Drosophila Mitotic Neuroblasts (Daul et al. 2010a) and Multicolor Fluorescence RNA In Situ Hybridization of Drosophila Brain Tissue (Daul et al. 2010b).

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Figure 1. Cell-fate markers in larval brain neuroblasts. Third instar larval brains stained with antibodies against the neuroblast markers Deadpan (Dpn; types I and II; green) and Asense (Ase; type I only; blue), and the cortical marker Discs large (Dlg; red). (A) Wild-type brain showing Dpn+ Ase+ type I neuroblasts (white arrowheads) and Dpn+ Ase- type II neuroblasts (black arrowheads). (B) An lgl; pins double mutant brain showing overproliferation of both type I and type II neuroblasts as determined by coexpression of Dpn and Ase. Anterior is to the top in all images. Scale bars, 20 µm. (Images courtesy of J. Haenfler, University of Michigan.)

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Figure 2. Cell-polarity markers in neuroblasts. (A) Metaphase wild-type neuroblasts display apical polar localization of aPKC (green) and basal polar localization of Miranda (Mira; blue). The mitotic spindle is visualized with anti--tubulin (Tub; red). (B) During telophase, apical proteins such as aPKC (green) are retained in the neuroblast (apical daughter), and basal proteins such as Mira (blue) are segregated into the ganglion mother cell (GMC; basal daughter). The mitotic spindle is visualized with anti--tubulin (red). (C) Cell polarity is disrupted in lgl; pins double mutants. aPKC (green) is localized uniformly around the cortex, displacing Mira to the cytoplasm (blue). Apical is oriented to the top, basal to the bottom in all images. Scale bars, 10 µm. (A, Images courtesy of C. Gamble, University of Michigan; C, images courtesy of J. Haenfler, University of Michigan.)

MATERIALS

Reagents

Antibodies (primary and secondary) of choice

 Block solution (D)

Drosophila larvae

 Fix solution

Glycerol (70%)

 PBSBTX

 PBSTX

ProLong Gold antifade mounting medium (Invitrogen)

Schneider’s insect medium (Sigma-Aldrich) (prechilled)

Equipment

Coverslips (22- x 22-mm [#1 thickness] and 24- x 40-mm)

Dissection dishes

Forceps (fine-tipped, two pairs)

Microcentrifuge tubes (0.5-mL)

Micropipettors and sterile tips

Microscope slides

Nutator mixer or rocker

Pipette with the tip cut off

METHOD

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 synchronizethem 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- x 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 beingdeformed by the 24- x 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 uprightposition. 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- x 40-mm coverslip over the samples and backfill the space between the slide and the coverslip by pipetting a small amountof mounting medium along the edge of the coverslip. 
  Backfilling will reduce the formation of air bubbles trapped in the slide.
  See Troubleshooting. 
  

TROUBLESHOOTING

Problem: The ventral nerve cord breaks off during dissection.

[Step 17]

Solution: Keeping the ventral nerve cord intact requires that you grasp the larva at the right place on its body. Holding the larva at a "sweet spot" near the fourth or fifth abdominal segment will typically allow clean dissection of the brain. Take care to gently break away attached tissues as you tear the head away from the body. The ventral cord is connected to the body by many axons and will likely break off if the head is carelessly pulled from the body.

Problem: There is poor signal-to-noise ratio.

[Step 19]

Solution: High levels of background staining can result from several steps in this protocol. Consider the following:

• 1. Ensure that all solutions are at the correct pH because high or low pH levels can negatively affect antibody binding. 
  
  

• 2. It is critical to use both primary and secondary antibodies at the appropriate dilution specific for each antibody. Test the specificity of secondary antibodies by staining a sample with secondary antibody alone. 
  
  

• 3. Thorough washing of samples is important for reducing backgroundsignals, particularly after incubation in primary antibodies. Place a small, fine pipette tip over a larger 1000-µL tip to help remove as much of the wash solutions as possible without losing or damaging the samples. However, note that excessive washing can also lead to weak signal strength. 
  
  

• 4. Antibodies can be sensitive to the duration and temperature of incubation. Anti-Dpn, for example, will typically yield cleaner staining when incubated for 3-4 h at room temperature than when incubated overnight at 4°C. Testing different incubation conditions might be necessary to determine the optimal conditions for a particular antibody.