Materials and Reagents

1. Coverslip stand for 5-10 coverslips (e.g., Wash-N-Dry, Diversified Biotech, catalog number: WSDR-1000 )
   
2. Gloves
   
3. A small plastic Petri dish or the cap of a 50 ml Falcon tube (~30 mm diameter)
   
4. 15 x 20 cm black plastic plate
   
5. High precision coverslips (e.g., Marienfeld Precision Cover Glass, 22 x 22 mm, Marienfeld-Superior, catalog number: 0107052 )
   
6. X-ray film (e.g., Amersham HyperfilmTM ECL, GE Healthcare, catalog number: 28906835 )
   
7. Double-sided adhesive tape (e.g., Tesa Doubleband Photostrip, Tesa, catalog number: 05338-00 )
   
8. Cheesecloth
   
9. A fly line or a cross that yields females of the appropriate genotype (see Note 1)
   
10. Dry, granular baker’s yeast (e.g., Lesaffre Saf-Instant^®)
    
11. Halocarbon oil (e.g., Voltalef 10S, VWR, catalog number: 24627.188 )
    
12. 100% ethanol (e.g., EMD Millipore, catalog number: 100983 )
    
13. ~5% dichlorodimethylsilane (DCDMS) in heptane (e.g., Silanization Solution, Sigma-Aldrich, catalog number: 85126 )
    
14. PIPES (e.g., Sigma-Aldrich, catalog number: P3768 )
    
15. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (e.g., EMD Millipore, catalog number: 105833 )
    
16. EGTA (e.g., Sigma-Aldrich, catalog number: E3889 )
    
17. HEPES (e.g., Sigma-Aldrich, catalog number: H3375 )
    
18. Potassium chloride (KCl) (e.g., EMD Millipore, catalog number: 104936 )
    
19. Dextran sulfate, MW ~10 kDa (e.g., Sigma-Aldrich, catalog number: D4911 )
    
20. Agar (e.g., from Pro-BIO)
    
21. Dry yeast (e.g., from Volk Klaus)
    
22. Soya powder (e.g., from Ruckemann)
    
23. Sirup (e.g., from Ruckemann)
    
24. Malt extract (e.g., from Baeko Rhei)
    
25. Corn powder (e.g., from Ruckemann)
    
26. Propionic acid (e.g., VWR, catalog number: ACRO447231000)
    Manufacturer: Acros Organics, catalog number: 447231000 .
    
27. Nipagin (e.g., Sigma-Aldrich, catalog number: H5501 )
    
28. BRB80 (see Recipes)
    
29. 1% injection buffer (1% IB) (see Recipes)
    
30. Cornmeal agar (see Recipes)
    

Equipment

1. Standard fly husbandry equipment (e.g., vials with cornmeal agar–see Recipes, 25 °C incubator with humidity controller, brushes, CO₂ station, etc.)
   
2. 3-9 well dissection plate (e.g., Corning, catalog number: 7220-85 )
   
3. Vacuum desiccator (e.g., SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F42025-0000 )
   
4. Dumont #5 forceps (e.g., Fine Science Tools, catalog number: 11252-20 )
   
5. Dumont #55 forceps (e.g., Fine Science Tools, catalog number: 11255-20 )
   
6. Custom made coverslip holder for microscopy (Figure 1)
   
   
   Figure 1. Vacuum desiccator loaded with the coverslip holder and the silane reservoir. After replacing the lid of the device, apply vacuum by opening the vacuum valve and the water tap. Once the vacuum is formed–i.e., you can lift the entire device by the lid–close the vacuum valve and the water tap.
   
   
7. Zooming stereomicroscope with 5-40x (or higher) magnification (e.g., Carl Zeiss, model: STEMI SV 11 )
   
8. Sharp tungsten needles with handles (e.g., Fine Science Tools, catalog numbers: 10130-20 and 26018-17 )
   
9. Total Internal Reflection Fluorescent (TIRF) microscope equipped with a high NA objective and a high sensitivity detector (Leica Microsystems, model: Leica AF7000 )
   Note: We use a Leica 7000 wide-field TIRF microscope with a 100x 1.46 NA oil immersion objective and a Photometrics Evolve Roper 512 EMCCD camera.
   

Software

1. Microscope controller software (e.g., Leica LASAF)
   
2. ImageJ/FIJI
   
3. Custom-made particle detector and tracker plug-in for ImageJ (available to download under https://github.com/Xaft/xs/blob/master/_xs.jar)
   
4. R (preferentially with RStudio), for data analysis
   

Procedure