4.8. Histology

For histopathological preparation 4% (wt/vol) formalin‐fixed paraffin‐embedded HNSCC tumors were cut to 4 μm sections, mounted on microscope glass slides, and heated overnight at 65°C in a drying oven. Following dehydration, slides were stained with hematoxylin and eosin (H&E), scanned by a Panoramic MIDI II scanner (3D Histech) and analyzed by a pathologist. Necrotic areas within treated tumors were morphologically evaluated. First, the AON was marked and was calculated in arbitrary units using the ImageJ and CaseViewer programs, after which the AON was calculated as a percentage of the entire tumor volume (AON%). Morphological characteristics of necrosis consisted of areas of atypical mitosis, lymphocytes, fibrin, acute inflammation, and tissue loss. Results are presented in AON%. Statistical analysis was carried out by GraphPad Prism 7.03 software, significance set at p = 0.05.