Cytokeratin 19

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Cytokeratin 19 is a protein expressed in epithelial cells. It is commonly used as a marker for the identification and characterization of epithelial cells and their derivatives in various applications, such as immunohistochemistry and flow cytometry.

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Anti-cytokeratin 19 (sc-25724, Anti‐Cytokeratin 19 antibody

7 protocols using cytokeratin 19

Western Blot Analysis of Cell Lysates

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Cell lysates were analyzed by SDS-PAGE and transferred onto 0.45 μM nitrocellulose membrane (Bio-Rad). Membranes were blocked using 5% low fat dry milk in TBST and probed with the respective primary antibodies. Proteins were detected using ECL Western Blotting Substrate (Thermo Scientific) and autoradiography. The protein loading was normalized using antibody to β-actin or GAPDH. The following antibodies were used in this study: Nanog, Vimentin, c-Met c-Myc, Sox2, Notch 1, pERK, ERK, c-Fos (Cell Signaling Technologies), cytokeratin19, cyclin D1 and β-actin (Santa Cruz Biotechnology).

Muhammad N., Bhattacharya S., Steele R., Phillips N, & Ray R.B. (2016). Involvement of c-Fos in the promotion of cancer stem-like cell properties in head and neck squamous cell carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(12), 3120-3128.

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Comprehensive Immunohistochemical Profiling

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Primary antibodies used for immunoblotting, co-immunofluorescence (co-IF), and immunohistochemistry: ARID1A/BAF250A (Cell Signaling Technologies, D2A8U), VIMENTIN (Cell Signaling Technologies, D21H3) for co-IF with E-CADHERIN and immunohistochemistry, E-CADHERIN (Cell Signaling Technologies, 4A2), Cytokeratin 19 (Santa Cruz Biotechnologies, A-3), KI-67 (MIB-1) (Dako), Cleaved CASPASE 3 (A175) (Cell Signaling Technologies, 5A1E), HSP90 alpha (Invitrogen, PA3–013), VIMENTIN (Cell Signaling Technologies, 5G3F10) for immunoblotting and co-IF with HSP90. ARID1B/BAF250B (Cell Signaling Technologies, E9J4T), PBRM1/BAF180 (Cell Signaling Technologies, D3F7O), BRM (Cell Signaling Technologies, D9E8B), BRG1 (Cell Signaling Technologies, A52), SMARCC1/BAF155 (Cell Signaling Technologies, D7F8S), SMARCC2/BAF170 (Cell Signaling Technologies, D8O9V), BAF60a (Santa Cruz Biotechnologies, 23), BAF53 (Santa Cruz Biotechnologies, C-7), SMARCB1/BAF47 (Cell Signaling Technologies, D8M1X). Chemical reagents: NVP-AUY-922 (LC Laboratories).

Tomihara H., Carbone F., Perelli L., Huang J.K., Soeung M., Rose J.L., Robinson F.S., Deribe Y.L., Feng N., Takeda M., Inoue A., Del Poggetto E., Deem A.K., Maitra A., Msaouel P., Tannir N.M., Draetta G.F., Viale A., Viale A., Heffernan T.P., Bristow C.A., Carugo A, & Genovese G. (2020). Loss of ARID1A promotes epithelial-mesenchymal transition and sensitizes pancreatic tumors to proteotoxic stress. Cancer research, 81(2), 332-343.

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Immunofluorescence Analysis of Tumor Tissues

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Frozen tissue samples of mice tumors and corresponding samples of normal liver tissue were sectioned into 5 μm frozen sections on a cryomicrotome (Leica, Buffalo Grove, IL), air-dried and stored at -80°C. Sections were fixed with 4% paraformaldehyde and permeabilized using Triton-X. Sections were subsequently blocked for 1 h at room temperature with calcium- and magnesium-free Dulbecco's phosphate buffered saline (PBS) containing 5% bovine serum albumin (BSA), and incubated with primary antibody for 12 h at 4°C. Antibodies were diluted in PBS containing 5% BSA. Primary antibodies and their dilutions were as follows: green fluorescent protein (GFP) (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), ST2 (1:50; Abcam, Cambridge, MA), cytokeratin 19 (1:50; Santa Cruz Biotechnology), and α-smooth muscle actin (α-SMA; 1:100, Abcam). After washing, slides were incubated with corresponding secondary antibodies in the dark for 1 h at room temperature, washed again, and mounted using Prolong Antifade with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Grand Island, NY) to visualize the nuclei. The slides were analyzed using fluorescent confocal microscopy equipped with an ultraviolet laser (LSM 780; Zeiss, Jena, Germany).

Yamada D., Rizvi S., Razumilava N., Bronk S.F., Davila J.I., Champion M.D., Borad M.J., Bezerra J.A., Chen X, & Gores G.J. (2015). IL-33 Facilitates Oncogene Induced Cholangiocarcinoma in Mice by an IL-6 Sensitive Mechanism. Hepatology (Baltimore, Md.), 61(5), 1627-1642.

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Extraction and Analysis of Se-PFPs

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The extraction and component analysis of Se-PFPs were performed as described previously [21 (link), 22 (link)]. Primary antibodies to Bax, Bcl-2, E-cadherin, N-cadherin, Cytokeratin 19, Vimentin, ZEB1, ZEB2, MMP-9, β-catenin, GSK-3β, CyclinD1, phosphorylated GSK-3β, phosphorylated β-catenin, cleaved caspase-3 and PARP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). α-tubulin and lamin A/C antibodies (cell signaling technology), MG132 was purchased from Sigma. HEY and SKOV3 cells were initially obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C under 5% CO₂.

Sun Q., Dong M., Wang Z., Wang C., Sheng D., Li Z., Huang D, & Yuan C. (2016). Selenium-enriched polysaccharides from Pyracantha fortuneana (Se-PFPs) inhibit the growth and invasive potential of ovarian cancer cells through inhibiting β-catenin signaling. Oncotarget, 7(19), 28369-28383.

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Immunofluorescent Staining of TM Explants

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Following fixation, collagen gel or whole mount TM explants were rinsed 2× in PBS. Permeabilization was performed for 15 mins with 0.5% TritonX-100 in PBS at RT with gentle shaking. Next, samples were rinsed three times with PBS for 5 min and blocked in 3% donkey serum in PBS for two hours. Explants were then incubated with Cytokeratin-19 (1:200; Santa Cruz Biotechnologies, Dallas, TX, USA) and Vimentin (1:200; Santa Cruz Biotechnologies, Dallas, TX, USA) primary antibodies in 1% donkey serum containing PBS at 4 °C overnight. Following three times rinsing and washing in PBS, anti-rabbit Cy3 (1:500) and anti-mouse-FITC (1:500) conjugated secondary antibodies (Jackson Immunoresearch Laboratories Inc., West Grove; PA, USA) were added and kept at 4 °C overnight with gentle shaking. Following incubation, explants were rinsed in PBS with 0.5% Tween-20 for 2 × 1 hours. Nuclei were counterstained with 1 μM DAPI in PBS. Finally, both, collagen and floating explants were carefully transferred to a microscopic slide and mounted. Images were captured utilizing Zeiss LSM 710 laser scanning confocal microscope (Zeiss, Jena, Germany) and Nikon Eclipse3 epifluorescent microscope (Nikon Corp., Tokyo, Japan), respectively. For image processing, Z-stack and tiles were interconnected using ImageJ software.

Bako P., Lippai B., Nagy J., Kramer S., Kaszas B., Tornoczki T, & Bock-Marquette I. (2023). Thymosin beta-4 – A potential tool in healing middle ear lesions in adult mammals. International immunopharmacology, 116, 109830.

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Immunofluorescence Staining of Brain Metastasis

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The cells of LLC-brain metastatic tumor (BrM) were seeded on coverslips, followed by incubation for 12 h at 37°C. The cells were fixed with 4% paraformaldehyde and then treated with 0.2% Triton X-100 for permeabilization, after which 5% bovine serum albumin in phosphate-buffered saline (PBS) was used for blocking. The cells were stained with antibodies against Cytokeratin 19 (dilution of 1:100; Santa Cruz Biotechnology, Inc., TX, USA) and then incubated with a secondary antibody conjugated with Alexa-fluorescence 594 (1:200 Invitrogen, CA, USA). After staining with 4′,6-diamidino-2-phenylindole, confocal laser scanning microscopy (LSM 880; Zeiss Co., Ltd., Oberkochen, Germany) was used for observation and analysis. In the immunofluorescence of the brain slice (50 μm) the primary antibodies used were as follows: HOXB9 (1:100 in dilution); CD31 (1:100; BD Biosciences, NJ, USA); Ki67 (1:100; Cell Signaling Technology, Inc., MA, USA).

Wei X.G., Bi K.W, & Li B. (2021). Phenotypic Plasticity Conferred by the Metastatic Microenvironment of the Brain Strengthens the Intracranial Tumorigenicity of Lung Tumor Cells. Frontiers in Oncology, 11, 637911.

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Immunohistochemical Profiling of Cholangiocyte Markers

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Detection of the cholangiocyte marker cytokeratin 19 (Santa Cruz Biotech., Santa Cruz, CA, USA, cat# sc-33111, RRID:AB_2234419), adhesion-marker E-Cadherin (Abcam, Berlin, Germany, cat# ab11512, RRID:AB_298118), tight-junction marker ZO-1 (Novus Biologicals, Bio-Techne GmbH, Wiesbaden, Germany, cat# NBP1-85047, RRID:AB_11023321), Ki67 (Abcam, cat# ab27619, RRID:AB_471081), SOX9 (Santa Cruz Biotech., cat# sc-20095, RRID:AB_661282), c-Jun (Cell Signaling Technology, Frankfurt am Main, Germany, cat# 9165, RRID:AB_2130165), and CD3 (Abcam, cat# ab16044, RRID:AB_443294) was performed as described [27 (link)].

Hahn L., Helmrich N., Herebian D., Mayatepek E., Drebber U., Domann E., Olejniczak S., Weigel M., Hain T., Rath T., Wirtz S., Mollenkopf H.J., Schmidt N., Ewers C., Baier A., Churin Y., Windhorst A., Weiskirchen R., Steinhoff U., Roeb E, & Roderfeld M. (2020). IL-13 as Target to Reduce Cholestasis and Dysbiosis in Abcb4 Knockout Mice. Cells, 9(9), 1949.

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