The Phagocytosis Assay: An Immunostaining Technique to Visualize Apoptotic Cell Engulfment in the Embryonic Cells of Drosophila melanogaster

Macrophages are specialized immune cells that clear the cells undergoing apoptosis - programmed cell death by phagocytosis.

To assess apoptotic cell engulfment, begin with a glass slide containing fixed Drosophila embryonic cells, including apoptotic bodies engulfed by hemocytes - invertebrate macrophages.

First, treat the cells with a blocking reagent that blocks the non-specific sites. Add primary antibodies that bind the croquemort receptors - phagocytic markers of hemocytes. Wash off the excess antibodies.

Add alkaline phosphatase-labeled secondary antibodies that bind to the primary antibodies. Now, add an appropriate chromogenic substrate solution on the slide, and incubate. During incubation, alkaline phosphatase reacts with the chromogenic substrate to yield purple-stained granules in the hemocytes.

Next, add a reaction mixture comprising terminal deoxynucleotidyl transferase, TdT enzyme, and digoxigenin-bound nucleotides. TdT catalyzes the attachment of digoxigenin-bound nucleotides to the exposed 3' OH ends of the fragmented DNA in the apoptotic bodies.

Next, add anti-digoxigenin antibodies conjugated to peroxidase reporter that bind to the nucleotides at the site of DNA damage. Treat the cells with a peroxidase-specific chromogenic substrate solution, and incubate. Peroxidase catalyzes the oxidation of the substrate, generating colored precipitates and staining the apoptotic bodies brown.

Finally, visualize the cells under a light microscope. The co-presence of purple granules and brown signals from the cells indicates hemocytes containing phagocytosed apoptotic bodies.

To immunostain the cells with anti-Croquemort antibody, first, serially soak the slide in methanol, then PBS supplemented with 0.2% Triton X-100, followed by PBS alone. Next, block the non-specific binding with 20 microliters of 5% whole swine serum in PBS + 0.2% Triton X-100, for 20 minutes at room temperature. Remove the excess blocking solution, and label the cells with 20 microliters of anti-Croquemort antiserum at 4 degrees Celsius overnight.

The next morning, wash the slide in PBS + Triton X-100 for five 10-minute incubations. After the last wash, rinse the slide in PBS for 10 minutes. Then, label the cells with 20 microliters of alkaline phosphatase-labeled anti-rat IgG at room temperature for 1 hour.

At the end of the incubation, wash the slide five times in PBS + Triton X-100 as demonstrated, followed by a 10-minute soak in buffer solution. Then, label the cells with phosphatase substrate solution, and observe the cells under a light microscope.

When strong purple signals appear in the hemocyte granules, remove the excess substrate, and soak the slide in buffer supplemented with EDTA for 5 to 10 minutes. At the end of the incubation, rinse the cells with two 5-minute PBS washes, and treat them with equilibration buffer for 10 minutes.

After removing the solution, label the cells with 20 microliters of terminal deoxynucleotidyl transferase solution at 37 degrees Celsius for 1 hour. Then, remove the solution from the slide, and soak the cells in 0.5 milliliters stop wash buffer in 17 milliliters of water, for 10 minutes. Next, rinse the cells with three 5-minute PBS washes, and label them with 20 microliters of anti-digoxigenin peroxidase for 30 minutes at room temperature.

At the end of the incubation, wash the cells four times in PBS, as demonstrated, and soak the slide in peroxidase substrate for 30-second increments until the apoptotic cells turn brown. When an optimal staining has been achieved, soak the slide in water to stop the peroxidase reaction.