Diaminobenzidine substrate kit

Specimens were cut into 3-µm-thick sections. An anti-ADP antibody (Progen Biotechnik GmbH, Heidelberg, Germany) was used as the primary antibody. Samples were then incubated with horseradish peroxidase-labeled goat anti-mouse secondary antibody (Histofine; Nichirei Biosciences, Tokyo, Japan). Immunoreactions were visualized by using a diaminobenzidine substrate kit (Nichirei Biosciences). ADP expression was said to be positive when granular or vacuolar cytoplasmic expression was observed in the tumor cells. Foamy macrophages in the specimen were used as a built-in positive control, according to a previous study [11 (link)].

Paraffin-embedded HGSOC blocks were cut into 3-μm-thick sections, after which sections were deparaffinized and rehydrated. Epitopes were then retrieved by means of heat-induced antigen retrieval—boiling the sections in a pressure cooker in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 20 min. The DO-7 mouse monoclonal anti-p53 antibody that was used for p53 immunohistochemical evaluation of ovarian carcinoma (21 (link)) was the primary antibody (1:100). Sections were then incubated with a horseradish peroxidase-labeled goat antimouse secondary antibody (Histofine; Nichirei Biosciences, Tokyo, Japan). Signals were detected by using a diaminobenzidine substrate kit (Nichirei Biosciences). A senior pathologist (Y.K.) and experienced oncologists (N.I. and K.I.) analyzed the specimens.