To follow the dynamics of apoptotic cell clearance in vivo two populations of cells must be labeled: phagocytic cells and apoptotic cells.

To mark phagocytic cell populations we use a Drosophila line containing the simu-cytGFP marker, which labels exclusively phagocytic cells in the embryo: macrophages, glia and ectoderm⁸. One can use different markers for phagocytic cells, including lines containing a hemocyte-specific Gal4 driver (crq-Gal4) or glia-specific Gal4 driver (repo-Gal4) and a genetically encoded fluorescent reporter under UAS control (uas-GFP).

To monitor apoptotic cells during apoptotic cell clearance we use different apoptosis/phagocytosis markers. Annexin V (Molecular Probes) serves as an early marker for apoptotic cells ¹⁰; Phiphilux (OncoImmunin) is a fluorogenic caspase-3 substrate, which is used as a later apoptosis marker, and LysoTracker (Molecular Probes) works as a phagosome marker. We inject these reagents using the microinjection system PicoPump PV 820.

1. Getting Ready

1. Heptane glue:
   Unroll double sided tape and put as much as you can in a scintillation vial, fill with heptane, seal the vial with Parafilm, and rock for 24 hr. Add heptane if the glue is too thick.
2. We allow the flies to lay on a pre-warmed grape juice agar plate + yeast paste for 2 hr, and then transfer the plate to 25 °C for 10-12 hr (depends on the desired stage) prior to microinjection and time lapse recordings of injected embryos. We collect embryos from the agar plates maintained at 25 °C, thus providing embryos from stage 15 or 16 of development.
3. Needle preparation:
   To prepare needles for injection, we use a ‘Sutter instrument’ needle puller and thin walled glass filaments (FHC Capillary tubing). The tip of the pooled capillary is broken to a diameter of 0.5-2 μm.
4. Coverslip with glue:
   Using a pipette tip disperse a drop of the heptane glue in one line in the middle of the coverslip. Let it dry for a few seconds. Prepare a few of these coverslips.

2. Embryo Preparation

1. Collect embryos from agar plates and transfer them to a cell strainer (SPL Life Sciences) using a clean paint brush and running water. The strainer is held over a beaker to collect waste water.
2. Wash embryos in the cell strainer using water until all the yeast paste is removed.
3. Dry out the excess water by wiping the outside of the strainer using Kimwipes.
4. Place the cell strainer in a clean Petri dish and add enough 50% bleach to cover embryos in the cell strainer. Dechorionate the embryos for 2 min occasionally (2-3 times) stirring them.
5. Rinse with water extensively until embryos loose the bleach odor.
6. Place the cell strainer into a clean Petri dish and cover the embryos with water to prevent drying up.
7. Before starting embryos setting, load 1 μl of the desired reagent into two needles (sometimes a needle can be clogged). It takes about 5 min for the liquid to reach the tip of the needle.
8. Cut a rectangular piece of grape juice agar and place it on a microscope slide. Transfer dechorionated embryos from the Petri dish to the agar piece using a clean paint brush.
9. Under a fluorescent dissection microscope select properly staged embryos expressing the GFP marker using a wet paint brush and place each embryo close to the edge of the agar piece in a row one after another (Figure 1A). The embryos should be placed with their ventral side up for imaging phagocytosis by glia in the Central Nervous System (CNS).
10. Set up about 10 embryos in one row.
11. Attach the embryos to a coverslip coated in the middle with a strip of the heptane glue (Figure 1B, C).
12. Place the coverslip in the dehydration chamber for about 5 min (this depends on the humidity of the room and the amount to be injected).
13. After drying up cover the embryos with halocarbon oil 700 (Sigma) to avoid further dehydration.
14. Place the coverslip on a microscope slide with the embryos facing up. You may put a drop of water on the slide to prevent moving of the coverslip. Embryos are now ready for injection. Injection location is typically on the lateral side in the middle of the embryo.

3. Embryo Injection

1. Attach the needle to a micromanipulator.
2. To break the needle tip put the edge of the coverslip in the field of view and adjust the needle to the same focal plane. Very carefully move the coverslip until it hits the needle tip and breaks it.
3. Focusing on the embryos, place the needle into the oil and make sure you get a liquid drop from the needle. It means the needle tip has been broken well.
4. Without touching the needle holder move the embryo into the needle and inject a drop into the embryo. Move the embryo away and proceed to the next one until all the embryos are injected.

4. Imaging

We image the embryos on an inverted confocal microscope with a 40X or 100X objective. We look for a nicely positioned embryo with the CNS in the middle, which shows strong GFP expression and a good labeling of apoptotic cells following injection.

For time lapse recordings of live embryos we usually choose 5 or 6 confocal slices (2 μm thick each). Afterwards, we make a projection of 3 slices (6 μm thickness) in order to observe whole cells.

We use markers for apoptotic cells which are stable and do not bleach easily. To avoid GFP bleaching we make recordings in intervals of 60 sec.

In some cases embryos may start rolling during the time of video recording. Therefore, we take a look once in a while at the recording in order to stop and start over if necessary.