Hematoxylin and eosin h e staining

Histologic sections (4-µm thick) of an oral cancer tissue microarray (catalogue number: OR481; US Biomax Inc.) were directly autoclaved in citrate buffer (pH 6.0; Agilent Technologies Inc.) for 20 min. Sections were then incubated with 1 µg/ml primary mAbs for 1 h at room temperature and treated using an Envision+ Kit (Agilent Technologies) for 30 min. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min, and sections were then counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation). hematoxylin and eosin (H&E) staining (FUJIFILM Wako Pure Chemical Corporation) was performed using consecutive tissue sections. Leica DMD108 (Leica Microsystems GmbH) was used to examine the sections and obtain images.

The formalin-fixed paraffin-embedded (FFPE) OSCC tissue microarray (Product Code: OR601c, US Biomax Inc., Rockville, MD, USA) and the esophageal tissue microarray (Product Code: BC02011, US Biomax Inc.) were deparaffinized in xylene (Sigma-Aldrich Corp.) and rehydrated. The FFPE OSCC tissue for peptide blocking assay was obtained from Tokyo Medical and Dental University [36 (link)]. The tissues were autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min for antigen retrieval. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C₄₄Mab-108 (10 μg/mL) and C₄₄Mab-46 (1 μg/mL), or without the primary antibody (control) for 1 h at room temperature and then treated with the EnVision+ Kit for mouse (Agilent Technologies Inc.) for 30 min. The color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min. Counterstaining was performed with hematoxylin (FUJIFILM Wako Pure Chemical Corporation). hematoxylin and eosin (HE) staining (FUJIFILM Wako Pure Chemical Corporation) was performed using consecutive tissue sections. Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to examine the sections and obtain images.