Confocal microscopic analysis of cocapping of LFA-1 and cholesterol. (A) Splenic T cells (upper row) and T28 cells (lower row) were stained with anti–LFA-1 and Alexa Fluor 568–conjugated goat anti-rat secondary antibody (red) and incubated at 37°C for 30 minutes to induce capping of LFA-1. The cells were then stained at 4°C for cholesterol with filipin (blue) and for GM1 with FITC-CTxB (green), fixed with formaldehyde, and analyzed by confocal microscopy. Images of midlevel sections of the cells are shown. Merge images are shown in the right-most columns. Results are representatives of multiple cells in 3 independent experiments. (B) Primary T cells were either untreated (rows 1, 3, and 5) or subjected to antibody cross-linking to induce capping of LFA-1 (row 2), Thy1 (row 4), or CD45 (row 6). The cells were then stained in 3 colors for cholesterol (blue), GM1 (green), and either LFA-1, Thy1, or CD45 (red) and analyzed by confocal microscopy as in panel A. (C) The T28 cell line was analyzed as in panel B. The images were captured with a × 60 objective lens, and the final magnifications in the figure are approximately × 1000 for primary T cells and × 500 for T28.