Colocalization of hypoxia and macrophages. Colocalization in human squamous carcinoma of the uterine cervix (A-D) and human PC3 (prostate carcinoma) xenografted tumors grown in nude mice (E-F). (A) Hypoxic areas (arrow), visualized by immunolabeling of the reductively activated hypoxic-specific marker pimonidazole (red), are observed at a distance from the disorganized blood vessels (BV). (B) In some areas of these tumors, CD68⁺ TAMs (brown) accumulate in stromal (S) rather than tumor (T) areas. (C-D) Colocalization of TAMS (brown) and hypoxia (red) showing TAM accumulation in avascular, perinecrotic, necrotic (N), and hypoxia areas. (E-F) Serial sections showing F4/80-positive (brown; panel F) TAMs in hypoxic, perinecrotic areas (red; panel E) of PC3 tumors. N indicates necrosis; and T, tumor. The microscope used was a Leitz Orthoplan. Magnification of panel A, × 100; panel B, × 400; panel C, × 160; panel D, × 160; panel E, × 160; panel F, × 160 (panels D-F, cropped images). Slight adjustments were made in brightness and contrast on each whole image. The temperature was room temperature. The imaging medium was DPX. Chromogens used were DAB (brown) 3,3-diaminobenzene and vector red (red) (Vector Laboratories, Burlingame, CA). The camera was a Fuji HC 3002 digital camera. Image processing was performed by Photograb 3002 (Windows platform).