Immunohistochemistry of macrophages double-labeled for CD68/CD86 or CD68/CD163. CD68-positive macrophages express CD86 and CD163. (A) Representative immunofluorescent images double-labeled for CD68/CD86 and 4′,6-diamidino-2-phenylindole (DAPI) at 1 day postinjury (DPI) and 5 DPI. CD68-positive macrophages expressing the M1 marker CD86 were located predominantly in necrotic tissue (white arrows). Similarly, macrophages expressing only CD86 were located in necrotic tissue (yellow arrows). Macrophages expressing only CD68 were located in non-necrotic perilesional tissue (red arrows). (B) Representative immunofluorescent images double-labeled for CD68/CD163 and DAPI at 1 DPI and 5 DPI. All CD68-positive macrophages were positive for CD163 to varying degrees (white arrows). The leftmost images in each row display low-magnification (4X) images (scale bars = 500 μm), with the rectangles indicating the selected areas of higher magnification (20X) (scale bars = 100 μm) shown in the three accompanying images to the right.