Preparation and Immunofluorescence Staining of the Trachea in Drosophila Larvae and Pupae

Materials and Reagents

1. 60 mm x 15 mm petri dish [i.e., Falcon^® Petri dish (Corning, catalog number: 351007 )]
   
2. Flat-bottom 4-well dish [i.e., Nunclon^® Δ Multidishes, 4 wells, flat bottom (Sigma-Aldrich, catalog number: D6789-1CS )]
   
3. Gold Seal^TM Rite-On^TM Frosted microslides (VWR International, Erie Scientific, catalog number: 3050 )
   
4. Micro cover glasses (coverslips), 22 x 22 mm Square No. 1 (VWR International, catalog number: 48366-067 )
   
5. Black electrical tape (i.e., 3M Scotch Super 33+ Vinyl Electrical Tape 0.75 in x 450 in)
   
6. Clear nail polish [i.e., crystal clear (Sally Hansen Hard as nails polish)]
   
7. Kimwipes^TM (4.4 x 8.4 in.) (Thermo Fisher Scientific, catalog number: 06-666 )
   
8. Austerlitz Insect Pins^® ,12 mm length x 0.10 mm diameter (Minutiens in stainless steel, size 0.10 mm) (Entomoravia)
   
9. 10 μl, 200 μl and 1,000 μl pipet tips (USA Scientific, TipOne, catalog number: 1111-3000 , 1111-0000 and 1111-2021 )
   
10. Disposable glass Pasteur pipettes (Corning, catalog number: 7095D-5x ) with 1 ml rubber bulbs (Sigma-Aldrich, catalog number: Z111589 )
    
11. Aluminum foil
    
12. Parafilm M^® All-Purpose laboratory film (2" x 250') (VWR International, Bemis Company, catalog number: PM992 )
    
13. Drosophila melanogaster larvae and pupae of desired genotype raised at 25 °C
    
14. Vials and bottles with closures [Fisherbrand^TM stock bottles (catalog number: AS117 ), Fisherbrand^TM cotton balls (catalog number: 22-456-880 ), Fisherbrand^TM Drosophila products, BuzzPlugs^TM (catalog number: AS277 ), Fisherbrand^TM Drosophila vials (catalog number: AS514 )] with Drosophila food (see Cold Spring Harbor Protocols, 2014)
    
15. Dow Corning SYLGARD^® 184 Silicone Elastomer Kit [184 SIL ELAST KIT 0.5 KG (Ellsworth Adhesives)]
    
16. 4% paraformaldehyde (PFA) diluted in PBS [i.e., 16% paraformaldehyde (VWR International, catalog number: 100503-916 ) diluted to 4% in PBS]
    
17. Primary antibody to stain protein of interest [i.e., Chicken-anti-GFP (Abcam, catalog number: ab13970 ) to stain tracheal-expressed GFP in ppk4-Gal4, UAS-GFP larvae and pupae]
    
18. Fluorescence conjugated secondary antibody to visualize and amplify primary antibody staining [i.e., Alexa488-conjugated Goat-anti-Chicken (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-11039 ) to stain the above anti-GFP primary antibody]
    
19. Normal serum from the same species as the secondary antibody [i.e., normal goat serum (Vector laboratories, catalog number: S-1000 )]
    
20. Vectashield^® mounting media (Vector Laboratories, catalog number: H-1000 )
    
21. NaCl
    
22. KCl
    
23. Na₂HPO₄
    
24. KH₂PO₄
    
25. 1x Phosphate-buffered saline (PBS) (see Recipes)
    
26. Triton^TM X-100 (Sigma-Aldrich, catalog number: X100 ) diluted to 0.1% in PBS (see Recipes)
    
27. Block solution (see Recipes)
    
28. DAPI staining solution (see Recipes)
    
29. Dissection dish (see Recipes)
    

Equipment

1. Incubator to house Drosophila set to 25 °C
   
2. Stereomicroscope with light source [i.e., Carl Zeiss^TM Stemi^TM 2000C with KL 300 LED Cold Light Source (120 V) (Thermo Fisher Scientific, catalog number: 12-070-284 )]
   
3. Dumont #5 mirror finish forceps biology tips/straight/inox/11 cm (Fine Science Tools, catalog number: 11252-23 )
   
4. Vannas spring scissors-straight/sharp/8 cm/3 mm cutting edge (Fine Science Tools, catalog number: 15000-00 )
   
5. Benchtop shaker (i.e., Bellco Glass 7744-06115 Mini Orbital Shaker)
   
6. Clay Adams^TM Nutator Mixer (BD, catalog number: 421105 )
   
7. P2, P20, P100, and P1000 Pipetman^® Pipettes (Gilson Scientific Ltd., catalog number: F144801 , F123600 , F123615 and F123602 )
   
8. Small watercolor paintbrush, round, size 0
   
9. Stainless steel spatula with a micro spoon end (Ted Pella Inc., catalog number: 13500 )
   

Procedure