Ncl l end

Manufactured by Leica

Sourced in Germany

The NCL-L-END is a laboratory equipment product manufactured by Leica. It is designed for specialized applications, but a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation.

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3 protocols using ncl l end

Immunohistochemical Profiling of OSCC

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For immunohistochemical staining, formalin-fixed, paraffin-embedded human OSCC tissue sections were used. The primary antibodies used were as follows: anti-NOTCH3, rabbit polyclonal, 1:200 (ab23426, Abcam, CA, USA); anti-SMA, mouse monoclonal, 1:100 (M0851, Dako, Glostrup, Sweden). Antigen retrieval was performed according to the manufacturer’s protocol. EnVision+ Dual Link (Dako) was used for secondary antibody, and coloration was conducted with diamino-benzidine substrate. For immunofluorescent double-staining, anti-NOTCH3, 1:200 (Abcam), SMA, 1:100 (mouse monoclonal, M0851, Dako or rabbit monoclonal, Clone SP171, Spring Bioscinece, CA, USA), CD34, 1:100 (mouse monoclonal, NCL-L-END, Leica Biosystems, Wetzlar, Germany) and Cytokeratin, 1:100 (mouse monoclonal, Clone AE1/AE3, M3515, Dako) were used as a primary antibody. For antigen retrieval, the sections were treated with Tris buffer (pH = 7.4) containing 0.1 mg/ml trypsin for 30 min at 37°C for CD34 antibody. Alexa Fluor 488 goat anti-rabbit IgG (A11008, Invitrogen, CA, USA) and Alexa Fluor 594 goat anti-mouse IgG (A11005, Invitrogen) were used as secondary antibody. DAPI was used for nuclear staining. The immunofluoroscent images were captured and analyzed using Axioskop2 plus microscope (Carl Zeiss, Jena, Germany).

Kayamori K., Katsube K.I., Sakamoto K., Ohyama Y., Hirai H., Yukimori A., Ohata Y., Akashi T., Saitoh M., Harada K., Harada H, & Yamaguchi A. (2016). NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma. PLoS ONE, 11(4), e0154112.

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Quantitative Analysis of Tumor Vasculature

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To characterize the tumor vasculature, the mean intratumoral MVD was quantitatively assessed according to immunohistochemical CD34 staining. We obtained formalin-fixed specimens embedded in paraffin of the 15 patients who underwent surgical tumor resection by thoracotomy. The specimens were cut into 4-μm slices and fixed to histology slides (X-tra, Leica Biosystems, Nussloch, Germany). After hematoxylin/eosin staining, the slides were stained with CD34 antibody (1:30, NCL-L END, Novocastra, Leica Biosystems, Nussloch, Germany) using an automated staining system (BenchMark XT, Ventana Medical Systems, Oro Valley, AZ, USA). The slides were scanned with a slide scanner (iScan Coreo, Ventana Medical Systems). A pathologist blinded to clinical and imaging data performed the histopathological analysis by visually counting the positive microvessels on the scanned images using public domain software (imageJ, http://rsbweb.nih.gov/ij).

Huellner M.W., Collen T.D., Gut P., Winterhalder R., Pauli C., Diebold J., Seifert B., Strobel K, & Veit-Haibach P. (2014). Multiparametric PET/CT-perfusion does not add significant additional information for initial staging in lung cancer compared with standard PET/CT. EJNMMI Research, 4, 6.

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Immunohistochemical Analysis of Tissue Samples

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FVMs were embedded in paraffin and cut into 3-μm sections. After removing the paraffin, the sections were rehydrated, blocked, and incubated with primary antibodies overnight. The next morning, the sections were incubated with the secondary antibodies for 30 min at room temperature. Nuclei were counterstained with Hoechst 33342 (H3570, 1:400 dilution; Life Technologies, Gaithersburg, MD). The primary antibodies were tenascin-C supplied by Juntendo University (20 μg/ml), α-smooth muscle actin (α-SMA, ab21027, 1:50 dilution; Abcam, Cambridge, MA), CD34 (NCL-L-END, 1:2000 dilution; Leica Biosystems, Newcastle, UK), glial fibrillary acidic protein (GFAP, Z0334, 1:500 dilution; Dako, Glostrup, Denmark), integrin α_V (AB1930, 1:1000 dilution; Millipore, Bedford, MA), smooth muscle myosin heavy chain SM1 (7599, 1:3000; YAMASA, Tokyo, Japan), and SM2 (7601, 1:400; YAMASA). The secondary antibodies were anti-mouse Alexa Fluor 488 (A11001, 1:800 dilution; Life Technologies), anti-rat Alexa Fluor 488 (A11006, 1:800 dilution; Life Technologies), anti-mouse Alexa Fluor 647 (A21247, 1:800 dilution; Life Technologies), anti-goat Alexa Fluor 647 (A21469, 1:800 dilution; Life Technologies), and anti-rabbit Alexa Fluor 647 (A21244: 1:200 dilution; Life Technologies). Sections were examined and photographed with a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).

Kobayashi Y., Yoshida S., Zhou Y., Nakama T., Ishikawa K., Arima M., Nakao S., Sassa Y., Takeda A., Hisatomi T., Ikeda Y., Matsuda A., Sonoda K.H, & Ishibashi T. (2016). Tenascin-C promotes angiogenesis in fibrovascular membranes in eyes with proliferative diabetic retinopathy. Molecular Vision, 22, 436-445.

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