Immunofluorescent staining of human HNSCC for αB-crystallin, pimonidazole-modified proteins and PAL-E. Shown is a representative biopsy section. The fluorescent grey scale images were binarized, resulting in black and white images for αB-crystallin staining (A) and pimonidazole-modified proteins staining, indicating the hypoxic areas (B). The merged image with αB-crystallin staining (assigned red), pimonidazole staining (assigned green) and PAL-E blood vessel staining (assigned blue) shows a substantial overlap between αB-crystallin and hypoxic regions. Hypoxic regions are mostly located in areas at greater distance from vessels (C).