Protein block serum free reagent

For immunofluorescence staining, the tissues were deparaffinized and endogenous peroxidases in the specimens were blocked using Peroxidase-Blocking Solution (DakoCytomation A/S, Agilent Technologies, Inc., Santa Clara, CA, USA) at room temperature for 5 min. Before immunohistochemical staining, we performed an antigen retrieval step by boiling the specimens in 10 mM citrate buffer (pH 6.0) using a microwave oven for 20 min. After incubation with Protein Block Serum-Free Reagent (DakoCytomation A/S) at room temperature for 5 min, the specimens were incubated with rabbit anti-α-SMA polyclonal antibody (1:100; Proteintech Group, Chicago, IL, USA) at room temperature for 60 min, and then washed. The specimens were then incubated with anti-rabbit immunoglobulin/TRITC (1:40, DakoCytomation A/S) at room temperature for 30 min. Nuclear staining and mounting were performed using VECTASHIELD^® Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The specimens were observed under a BZ-X700 fluorescence microscope (KEYENCE, Tokyo, Japan), and α-SMA fluorescence intensity was measured as the average intensity per unit area along the aorta using BZ-X800 Analyzer version 1.1.1.8 software (KEYENCE, Tokyo, Japan).

Peroxidase conjugated ABC reagent, Vectastain Elite ABC standard kit was from Vecter Laboratories, Inc. (Burlingame, CA, USA). Antihuman AGP rabbit serum and Protein Block Serum-Free Reagent were from Dako (Carpinteria, CA, USA). Peroxidase conjugated anti-human AGP and Universal HIER antigen retrieval reagent were from Abcam (Cambridge, UK). KPL SureBlue TMB Microwell Peroxidase Substrate was from Sera Care Life Sciences (Milford, MA, USA). Anti-PD-L1 and SignalStain Boost IHC Detection Reagent were obtained from Cell Signaling Technology (Danvers, MA, USA). N-Histofine High Stain HRP (Multi) was from Nichirei Biosciences Inc. (Tokyo, Japan). PNGase F was from Roche Applied Science (Indianapolis, USA). BlotGlyco was obtained from Sumitomo Bakelite, Co. (Tokyo, Japan). Neuraminidase (Arthrobacter ureafaciens, 1U/ml) was purchased from Nacalai Tesque (Kyoto, Japan). Biotinylated Aleuria aurantia lectin (AAL) was kindly provided by Prof. Naohisa Kochibe, Gunma University. Tumor-associated antigens in serum samples measured in this study were carcinoembryonic antigen (CEA) and squamous cell carcinoma (SCC). Levels of each antigen were determined by an ELISA using the Cobas system (Roche for CEA) and the ARCHITECT system (Abbot for SCC) and standard cut-off values were set at 5.0 ng/ml for CEA and 1.5 ng/ml for SCC, respectively.

Immunohistochemical staining was performed using 5-μm-thick sections. All sections were incubated at 60 °C for 60 min, deparaffinized in xylene, rehydrated, and incubated with fresh 0.3% hydrogen peroxide in 100% methanol for 30 min at room temperature to block endogenous peroxidase activity. After the specimens were rehydrated through a graded ethanol series, antigen retrieval was performed in an Immunosaver (Nissin EM, Tokyo, Japan) at 98–100 °C for 60 min, and sections were passively cooled to room temperature. After the sections were rinsed in 0.1 M of phosphate-buffered saline (pH 7.4), non-specific binding sites were blocked via incubation with Protein Block Serum-Free Reagent (Dako, Carpenteria, CA, USA) for 30 min. The sections were then incubated with D2-40 antibody (Dako) at a dilution of 1:200 overnight at 4 °C and at room temperature for 30 min. The reactions were visualized using a Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. The chromogen 3,3′-diaminobenzidine tetrahydrochloride was applied as a 0.02% solution in 50 mM OF ammonium acetate-citrate acid buffer (pH 6.0) containing 0.005% hydrogen peroxide. The sections were lightly counterstained with hematoxylin, and then mounted. Negative controls were incubated without the primary antibody, and no detectable staining was evident.