Immunohistochemical Analysis of Tumor Markers

NB specimens were fixed for 24–48 hr in 10% neutral buffered formalin (Bio Optica) and paraffin embedded. For histological analysis, 4 μm thick serial sections were stained with Harris’ hematoxylin and 1% eosin (Bio Optica). For immunohistochemistry experiments, serial sections were treated in a microwave oven three times for 5 min in citrate buffer (pH 6.0) at 750 W, then subjected to 3% H₂O₂ for 15 minutes and saturated with 20% goat serum in PBS-T, for 1 hr at 37°C. Samples were then incubated overnight at 4°C in a humidified chamber with the specific primary antibody diluted in PBS-T as follows: anti-VEGFA (1:50), anti-PHD3 (1:100), anti-PDK1 (1:100), anti-HIF-1α (1:150), and anti-N-Myc (1:200). Negative controls were made with PBS-T alone. After incubations with the biotinylated secondary antibody and the HRP-streptavidin (LSAB2 System-HRP kit, Dako), the reactions were developed and visualized as described above. The slides, which were blinded for specimen identification, were evaluated by a pathologist in a randomized order using a bright field microscope. Specimens were evaluated for the percentage of positive cells and for the staining intensity. The percentage of positive cells was confirmed by ImmunoRatio, a free, automated web-based image analysis application for scoring immune-stained slides [37 (link)]. (https://dx.doi.org/10.17504/protocols.io.jtxcnpn)