Envision kit for mouse

FFPE sections of OSCC tissue array (OR601c) and colorectal carcinoma tissue array (CO483a) were purchased from US Biomax Inc. (Rockville, MD, USA). The tissue arrays were autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C₄₄Mab-6 (1 μg/mL) and C₄₄Mab-46 (1 μg/mL) for 1 h at room temperature. The sections were further incubated with the EnVision+ Kit for mouse (Agilent Technologies Inc.) for 30 min. Then, a chromogenic reaction using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) was conducted. Hematoxylin (FUJIFILM Wako Pure Chemical Corporation) was used for the counterstaining. To examine the sections and obtain images, we used Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany).

Paraffin-embedded tissue sections of a breast cancer tissue array (Cat#T8235721-5, Lot#B104066; BioChain, San Francisco, CA, USA) were autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), tissue sections were incubated with TrMab-29 (5 μg/mL) for 1 h at room temperature and then treated with the EnVision + Kit for mouse (Agilent Technologies Inc.) for 30 min. 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 & 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.

Paraffin−embedded ESCC tissue microarray (Product Code: BC02011, US Biomax Inc., Rockville, MD, USA) were deparaffinized in xylene and rehydrated. Then, they were autoclaved in citrate buffer (pH 6.0; Agilent Technologies Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), sections were incubated with C₄₄Mab−46 (5 μg/mL) for 1h at room temperature and then treated with the EnVision+ Kit for mouse (Agilent Technologies Inc.) for 30 min. 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.

One formalin-fixed paraffin-embedded (FFPE) oral SCC tissue was obtained from Tokyo Medical and Dental University [47 (link)]. FFPE sections of pancreatic carcinoma tissue arrays (Catalog number: PA241c and PA484) were purchased from US Biomax Inc. (Rockville, MD, USA). Pancreas adenocarcinoma tissue microarray with adjacent normal pancreas tissue (PA241c) contains 6 cases of pancreas adenocarcinoma with matched adjacent normal pancreas tissue, with quadruple cores per case. One oral SCC tissue was autoclaved in citrate buffer (pH 6.0; Nichirei biosciences, Inc., Tokyo, Japan), and pancreatic carcinoma tissue arrays were autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C₄₄Mab-3 (1 μg/mL) and C₄₄Mab-46 (1 μg/mL) for 1 h at room temperature. Then, the sections were incubated 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.). Hematoxylin (FUJIFILM Wako Pure Chemical Corporation) was used for the counterstaining. A Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to examine the sections and obtain images.

One formalin-fixed paraffin-embedded (FFPE) tissue sample from an oropharyngeal squamous cell carcinoma patient who underwent surgery at Sendai Medical Center was used for this study (13 (link)). Written informed consent was obtained from the patient for sample procurement and subsequent data analyses. Tissue microarray (CC00-10-001) including lymphomas, normal lymph node, and normal thyroid was purchased from Cybrdi, Inc.
The 4-µm thick paraffin-embedded tissue sections were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with the SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.), tissue sections were incubated with C₂₀Mab-11 (5 µg/ml) for 1 h at room temperature and treated with the Envision+ Kit for mouse (Agilent Technologies, Inc.) for 30 min. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

The formalin-fixed paraffin-embedded (FFPE) oral SCC tissues were obtained as described previously [56 (link)]. We purchased a colorectal carcinoma tissue array (CO483a) from US Biomax Inc. (Rockville, MD, USA). We used a cat rectum paraffin tissue section (Zyagen; FP-312) as a negative tissue control [57 (link)]. The sections were autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), we incubated the tissue sections with C₄₄Mab-1 (1 μg/mL) and C₄₄Mab-46 (1 μg/mL) for 1 h. For isotype control, we used PMab-44 (mouse IgG₁), an anti-bovine PDPN mAb [58 (link)]. The peptide blocking assay was performed as described previously [30 (link)]. The sections were further treated with the EnVision+ Kit for mouse (Agilent Technologies Inc.) for 30 min at room temperature. The chromogenic reaction was conducted using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.). The counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation). To examine the sections and obtain images, we used Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany).

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.