I view dab universal kit

Chamber slides were used for staining cells (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Medium (1 ml) containing 1×10⁴ MIA PaCa-2 cells was added to the chamber slides. Following 24-h incubation, 0.1–1.0 mM MU dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemicals Industries, Ltd.) was added, ensuring that the concentration of DMSO in the medium did not exceed 0.1%. Upon 48-h incubation, cells were washed twice with PBS (2 ml/wash), fixed with 4% paraformaldehyde buffered with PBS at room temperature for 2 h and rinsed twice with PBS. Following antigen retrieval, tissue samples were incubated for 32 min at 37°C with 2.5 µg/ml biotinylated HA binding protein (HABP; catalog no., BC41; Hokudo Co., Ltd., Osaka, Japan). Streptavidin horseradish peroxidase conjugate and 3,3-diaminobenzidine (DAB) from the iVIEW DAB universal kit (Ventana Medical Systems, Inc., Tucson, AZ, USA) was used to visualize the results. Negative controls were stained with HABP following digestion of the tissue sections with 3.0 U/ml Streptomyces hyalurolyticus hyaluronidase (26 (link)).

Paraffin sections of 4-µm thickness were immunostained with the AE1/3 antibody (PCK26, cocktail antibody, Ventana; Roche Tissue Diagnostics Japan; cat. no. 760-2595) and the podoplanin antibody (clone D2-40, Dako Agilent Technologies, Inc.; cat. no. M3619), respectively, using an automated immunostainer (VENTANA BenchMark ULTRA system; Ventana Medical Systems, Inc.) according to the manufacturers' protocol. The incubation with secondary antibodies and detection were carried out using the I–VIEW DAB Universal kit (Ventana Medical Systems, Inc.; cat. no. 760-041) and Endogenous Biotin Blocking kit (Ventana Medical Systems, Inc.; cat. no. 760-050).
Podoplanin expression in budding cells, which was confirmed by AE1/3 expression, was evaluated independently by the same authors mentioned above (HM and NK). The results were scored from 0 to 3 based on the intensity of the staining at the membrane or in the cytoplasm: 0, no reactivity; +1, weak; +2, moderate; and 3, marked, that is, overexpression (Fig. 2). For analysis, the results were divided into 2 groups: negative (score 0) and positive (from +1 to +3). The ly of the tumor tissue was evaluated using hematoxylin and eosin staining or podoplanin staining for detecting lymphatic vessels (negative; positive).

The engrafted tumors were fixed with 10% phosphate-buffered formalin, and paraffin-embedded sections were stained using hematoxylin and eosin. Anti-human vimentin (Clone V9; #M0725; Dako, Glostrup, Denmark) and anti-human pancytokeratin (AE1/AE3; #M3515; Dako) antibodies were used for the confirmation of human cell-derived tumors. A staining without primary antibodies was also prepared as a control for nonspecific effects of primary antibodies. Secondary antibodies (I-VIEW DAB Universal Kit, #518100032) were purchased from Roche Diagnostics KK (Tokyo, Japan).

Tumor tissues were fixed using 10% buffered formalin. Serial 10-μm-thick sections were prepared from formalin-fixed, paraffin-embedded (FFPE) tissues. Sections were stained with Hematoxylin and Eosin and reviewed by a pathologist to determine the tumor location. Immunohistochemistry of chromogranin-A (CgA), CD56, neuron-specific enolase (NSE), synaptophysin (SYN), neurofilament, and S-100 was used for the routine diagnosis of PPGL. In addition, Fontana-Masson stain was used to detect the accumulation of melanin. To detect TH, DDC, DBH, PNMT, and succinate dehydrogenase complex, subunit B (SDHB) protein expression, the sections were incubated with anti-TH, DDC, DBH, PNMT, and SDHB antibody, respectively, at 4°C overnight after blocking with Protein Block Serum-Free (DAKO, Tokyo, Japan) with partial reference to previous articles (9 (link)). The details of each antibody are described in Supplementary material 1. Subsequently, the Ventana BenchiMark ULTRA fully automated immunostaining system (Roche Diagnostics, Basel, Switzerland) was used with the I-VIEWDAB universal kit (Roche Diagnostics) containing secondary antibodies (biotinylated mouse anti-goat IgG antibody, biotinylated mouse anti-goat IgM antibody, and biotinylated rabbit anti-goat IgG antibody), avidin-horseradish peroxidase, and 3,3′-diaminobenzidine.

For histological examination, extrahepatic bile duct carcinoma specimens were routinely fixed with formalin, embedded in paraffin, sectioned to a thickness of 4-µm, and mounted on saline-coated glass slides. Immunohistochemical examination was performed on deparaffinized sections using the standard avidin-biotin-peroxidase complex method with a BenchMark XT automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). The different phenotypes were investigated for mucin (MUC) expression using primary antibodies against MUC1 (#NCL-MUC-1, dilution, 1:50; clone Ma696), MUC2 (#NCL-MUC-2, dilution, 1:50; clone Ccp), MUC5AC (#NCL-MUC-5AC, dilution, 1:100; clone CLH2) and MUC6 (#NCL-MUC-6, dilution, 1:100; clone CLH5), all purchased from Novocastra (Leica Biosystems, Newcastle, UK). After washing in PBS three times, secondary immunostaining was performed with an i-VIEW DAB Universal Kit (Roche Diagnostics, Tokyo, Japan) for 28 min at 42°C.