Immunohistochemical Analysis of ING5 Protein Expression in Tumor Xenografts

Animal experiments were performed as previously described [21 (link)]. ING5 protein expression was measured using immunohistochemical analysis on 5-mm slices of formalin-fixed paraffin-embedded tumor xenografts in nude mice. To avoid inter-treatment bias, the tissue slides from all six groups were made on a single slide and subjected to the same immunostaining simultaneously. Antigens were retrieved by pretreating dewaxed sections in a microwave oven at 750 W for 5 min in a citrate buffer (pH 6) processed with the Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy). The enzymatic activities were developed using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. Following counterstaining with Mayer's hematoxylin (Invitrogen), slides were mounted in aqueous mounting medium (glycergel, Dako). Pictures were taken using a LEICA DM 4000B microscope, while the relative level of each protein was calculated using LEICA software. The percentage of the mock- over the chemotherapeutic-treated tumors was calculated and plotted.