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