Bs966m

Immunohistochemical staining for B7-H4 was performed with an anti–B7-H4 rabbit monoclonal antibody (D1M8I, 1:200; Cell Signaling, Danvers, MA) as per the manufacturer's recommendations. The patients’ formalin-fixed, paraffin-embedded tissue specimens (3-μm-thick sections) were deparaffinized, exposed to Peroxidazed 1 (PX968; Biocare Medical, Concord, CA, USA) for 5 minutes to decrease background staining, and then immersed in a universal decloaker (UD1000M; Biocare Medical) and placed in a pressure cooker at 121°C for 5 minutes. Next, the sections were blocked using blocking reagent (BS966M; Biocare Medical) for 30 minutes, incubated with primary antibody overnight at 4°C, incubated with the biotinylated secondary antibody (GU600H; Biocare Medical) for 10 minutes, and incubated with Streptavidin HRP Label (HP604; Biocare Medical) for 10 minutes. Finally, the sections were stained with 3,3′-diaminobenzidine chromogen (DB801L; Biocare Medical) and then counterstained with hematoxylin.
The pattern of B7-H4 immunohistochemical staining was recorded using a 4-score grading system (Figure 1): Score 0, no staining/negative; Score 1, apical pattern; Score 2, mixed apical and circumferential membranous staining with circumferential membranous staining in <10% of tumor cells; and Score 3, circumferential membranous staining in ≥10% of tumor cells.