Immunohistochemical Analysis of TROP2 Expression

Immunohistochemical staining was performed as reported previously with slight modification [26 (link),27 (link),28 (link)]. Surgically obtained samples were fixed in 10% buffered formalin for 24 h and embedded in paraffin. The formalin-fixed paraffin-embedded tissues were cut into 4 μm-thick sections. TROP2 antigen was retrieved using Heat Processor Solution pH 9 (Nichirei Biosciences, Tokyo, Japan) via heating at 100 °C for 40 min. The sections were incubated with rabbit anti-human TROP2 (1:1000, ab214488; Abcam, Cambridge, UK) for 90 min at room temperature followed by incubation with N-Histofine Simple Stain MAX-PO MULTI (724152; Nichirei Biosciences) for 30 min at room temperature. Immunoreactions were detected using 3,3′-diaminobenzidine tetrahydrochloride (725191; Nichirei Biosciences) as a chromogen (exposure for 10 min), and specimens were counterstained using hematoxylin.