Materials and reagents

Biological materials

The imaging approaches described in this protocol assume that a fly line is used where the protein(s) of interest are fluorescently tagged. Wherever available, lines expressing fluorescently tagged proteins under the control of the endogenous promoter in a mutant background are selected, as these provide expression levels close to endogenous, confirmed by western blotting. The fluorescent protein selected depends on experimental requirements. For two-color experiments, eGFP, NeonGreen [30], or StayGold [31] (the latter generally more photostable) are typically combined with mCherry, a relatively bright and stable red fluorophore [32,33].

Reagents

1. Instant dried yeast (Fermipan red)

2. Bacteriological agar (VWR, catalog number: 84609)

3. Sucrose (Sigma-Aldrich, catalog number: RDD023)

4. Apple or cranberry juice

5. Heptane (Sigma-Aldrich, catalog number: 246654)

6. Voltalef H10S PCTFE oil ARKEMA (Samaro, catalog number: AK6208)

7. Immersion oil for 3D-SIM imaging on OMX-Blaze, DV Immersion Oil Kit containing refractive index oil 1.514 (GE Healthcare, catalog number: 29163068)

8. Immersion oil with a refractive index of 1.518 for spinning disc confocal (ImmersolT 518 F, Carl Zeiss, catalog number: 433802-9010-000)

Solutions

1. Yeast paste (see Recipes)

2. Fruit juice agar plates (see Recipes)

3. Heptane glue (see Recipes)

Recipes

1. Yeast paste

Mix instant dried yeast with water (approximately 10 g of yeast in 12 mL of water) in a 100 mL beaker to achieve a thick, creamy consistency.

2. Fruit juice agar plates

a. Boil 9 g of agar in 300 mL of distilled water and, separately, 10 g of sucrose in 100 mL of apple juice (or cranberry juice).

b. Mix together the agar and sugar solutions once fully dissolved.

c. Pour the mix into Petri dishes of a size designed to fit snugly into the bottom of the fly collection chamber (typically a diameter of 50 mm and height of 20.3 mm) to a depth of ~15 mm. Leave to cool and dry at room temperature for ~1 h before use. Plates can be stored at 4 °C for 2–3 days. Before use, add a button-sized drop of yeast paste to each plate and leave to dry. The yeast paste needs to dry to prevent the flies from getting stuck in the liquid yeast.

3. Heptane glue

a. Fill a 50 mL conical tube with double-sided sticky tape (remove the release liner and tightly pack approximately 1 m of tape into the tube) and add 20 mL of heptane. Leave the tube rotating overnight.

b. Remove the tape and centrifuge the heptane glue at 15,700 RCF to remove solids. The glue can be kept indefinitely at RT in an Eppendorf tube sealed with Parafilm.

Laboratory supplies

1. Petri dishes, 50 mm diameter (Fisher Scientific, catalog number: 10655821)

2. Glass-bottom 35 mm dishes (MatTek, catalog number: P35G-1.5-14-C)

3. Paintbrush (size 3)

4. Fine forceps (Dumont No. 5)

5. Microscope slides (Appleton Woods, catalog number: MS529)

6. Double-sided sticky tape (Scotch 665 Double-Sided Tape)

Note: The adhesion of some brands of double-sided sticky tape is too strong to dechorionate the embryos successfully. Scotch tape is particularly well-suited for dechorionation.

7. Sticky tape (single-sided) for holding the fruit juice agar plate onto the fly collection chamber (Starlabs ID Tape 55 m long × 19.0 mm wide, catalog number: E9055-1913)

Equipment

1. Fly collection chamber: This is typically a Perspex cylinder with one end sealed with a fine wire mesh (so that flies can breathe but not escape) (Flystuff Inc., catalog numbers: 59-100 or 59-101)

2. Humidified incubator at 25 °C in a reverse day light cycle to optimize the embryo collection during standard laboratory working hours

3. Binocular dissecting microscope (Nikon, model: SMZ800)

4. Rotator mixer (Stuart, model: SB3)

5. Confocal microscope with FRAP unit

Note: The example shown in Figure 3A was acquired using a Perkin Elmer Spinning disc system running Volocity 6.3 software mounted on a Zeiss Axiovert microscope with a 63×/1.4 NA oil immersion objective lens (60×/1.3 NA Silicone immersion oil lenses are also suitable). This system is equipped with a Hamamatsu Orca ER CCD camera and 488 and 568 nm laser lines. Comparable FRAP experiments can be performed on many other confocal systems.

6. 3D-SIM microscope with blaze unit

Note: The example shown in Figure 4A–E was acquired using a DeltaVision OMX V3 Blaze microscope (GE Healthcare), fitted with a 60×/1.42 NA UPlanSApo oil immersion objective lens. This system is fitted with PCO Edge 5.5 sCMOS cameras and 488 and 593 nm laser lines. Other SIM systems are suitable for similar experiments.

Software and datasets

1. Volocity (Perkin Elmer)

2. SoftWoRx (GE Healthcare)

3. ImageJ/FIJI (http://fiji.sc/) [34]

4. SIMcheck plugin (http://downloads.micron.ox.ac.uk/fiji_update/SIMcheck/) [35]

5. Radial Profile plugin (https://imagej.net/ij/plugins/radial-profile.html)

Procedure