In vivo tracking of erythrocytes. (A) Macrophages were visualized by F4/80 staining and incorporated erythrocytes, by prior PKH26 labeling. Compared with the wt organ, F4/80 and PKH26 staining is decreased in the tg6 spleen. The most intense phagocytosis of erythrocytes was found in the marginal zone (arrowheads) between white (w) and red (r) pulp. In contrast, F4/80 as well as PKH26 staining was dramatically increased in the tg6 liver (same magnification of all images; bar represents 50 μm; v indicates central vein). (B) Illustration of the quantification procedure of the PKH26 fluorescent area using image analysis. Tissue area was defined as the total image area minus the cross-sectional area of all vessels (higher optical density compared with the tissue, marked red after image processing). The PKH26-stained area is evident from a much lower optical density compared with the tissue (marked green after image processing). (C) Compared with wt, the PKH26-positive area per tissue area was about 22 times smaller in the transgenic spleen but approximately 150 times larger in the liver of tg6 mice. Means (± SD) of 10 analyzed images of liver and spleen each of 4 animals of each line; ***P < .001.