Confocal laser scanning microscopy images of immunostained peripheral blood eosinophils of asthmatic subjects.
Representative images of human eosinophils stained with TRITC-conjugated secondary antibody (red) to detect the MBP as well as RANTES immunoreactivity, or BODIPY-FL–conjugated secondary antibody (green) to detect immunoreactivity against CD63. (A) Resting peripheral blood eosinophils labeled with BODIPY-FL indicating CD63 immunoreactivity. (B) Highly purified crystalloid granules immunostained with anti-CD63. (C) Higher magnification of a combined image of eosinophil crystalloid granules immunostained for MBP (TRITC) indicating core-associated immunoreactivity and CD63 indicating matrix-associated CD63 immunoreactivity. (D) Combined image of double immunofluorescence (MBP and CD63) staining of resting eosinophils; red (TRITC) represents immunoreactivity against MBP, and green (BODIPY-FL) is indicative of CD63 immunoreactivity. (E) Combined image of double immunofluorescence (RANTES and CD63) staining of resting eosinophils; TRITC detected RANTES, and BODIPY-FL detected CD63. (F-H) Double immunofluorescence (MBP and CD63) staining of IFN-γ–stimulated eosinophils (time = 10 minutes): MBP immunostaining (F), CD63 immunostaining (G), and combined image (H). (I-K) Double immunofluorescence (MBP and CD63) staining of C5a/CB-stimulated eosinophils (time = 10 minutes): MBP immunostaining (I), CD63 immunostaining (J), and combined image (K). (L) Combined image of isotype control for CD63 and MBP immunostaining. (M-O) Double immunofluorescence (MBP and CD63) staining of IL-3/IL-5/GM-CSF–stimulated eosinophils (time = 10 minutes): MBP immunostaining (M), CD63 immunostaining (N), and combined image (O). (P) Combined image of double immunofluorescence (MBP and CD63) staining of IFN-γ–stimulated eosinophils preincubated with genistein (10−6 M), which inhibited the IFN-γ–induced translocation of CD63. (Q-T) Double immunofluorescence (RANTES and CD63) staining of IFN-γ–stimulated eosinophils: RANTES immunostaining (Q), CD63 immunostaining (R), combined image (S), and DIC image of the same cell (T). Original magnification is × 630 for all images.
