Representative photomicrographs (×400) showing apoptotic eosinophil engulfment by unstimulated (A) SAEC. Eosinophils can be seen inside SAEC as peroxidase-positive dark staining cells. Cytospins were also prepared from SAEC that had ingested aged eosinophils and were removed from their wells by trypsin digestion. Under these conditions (B), OPD-stained apoptotic eosinophils can be seen inside the SAEC, with no eosinophils present on the portions of the slide from which epithelial cells were absent. (C) Representative fluorescent micrograph (×1,000) showing SAEC permeabilized before ingested eosinophils were stained with the anti-MBP MoAb BMK-13, followed by antimouse IgG-FITC. Bright green staining ingested eosinophils can be clearly seen inside the SAEC. (D) Apoptotic eosinophils were also stained with a CD9 MoAb conjugated to PE (CD9-PE) and allowed to interact with SAEC that had been stained with anti-CD44 MoAb and antimouse IgG-FITC. Both cell types were immunostained before the interaction assay (30 minutes) and visualized with a fluorescent microscope. The epithelial cell displays CD44⁺ green fluorescence. Two apoptotic eosinophils can be seen, both of which were stained with CD9-PE before the interaction assay. The PE-positive eosinophil (top) is interacting with the SAEC membrane but is not yet completely engulfed and therefore shows red fluorescence. The other (bottom) is within a phagosome inside the SAEC and is thus surrounded by a CD44⁺ membrane and therefore exhibits green fluorescence.