Cytokeratin ae1 ae3

Manufactured by Agilent Technologies

Sourced in United Kingdom, Denmark, United States

Cytokeratin AE1/AE3 is a lab equipment product used for the identification and classification of epithelial cells. It is a cocktail of two monoclonal antibodies that recognize a broad spectrum of cytokeratins, which are proteins found in the cytoskeleton of epithelial cells. This product is commonly used in immunohistochemistry and immunocytochemistry applications to detect the presence and distribution of epithelial cells in tissue samples.

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13 protocols using cytokeratin ae1 ae3

Immunohistochemical Analysis of Tumor Samples

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Tumors sections (4 μM-thick) were stained for H&E, Cytokeratin AE1/AE3 (Cat#M3515, Agilent, CA), IL8 (Cat#ab84995, Abcam, MA), Ki67 (Cat#Ab16667, Abcam) or Caspase-3 (Cat#Ab32351, Abcam) according to the manufacturer’s protocol, and evaluated using a Leica Microscope.

Alt E.U., Wörner P.M., Pfnür A., Ochoa J.E., Schächtele D.J., Barabadi Z., Lang L.M., Srivastav S., Burow M.E., Chandrasekar B, & Izadpanah R. (2020). Targeting TRAF3IP2, Compared to Rab27, is More Effective in Suppressing the Development and Metastasis of Breast Cancer. Scientific Reports, 10, 8834.

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Intraoperative Lymph Node Staging in Cancer

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All the SNs were subjected to intraoperative pathological evaluation. All the other LNs, including the non-radioactive and non-fluorescent LNs, were considered non-SNs. The non-SNs were divided longitudinally into 2 specimens, and a single representative cross section was stained with haematoxylin-eosin (HE) for a final postoperative diagnosis. The SNs were cut into 2-mm blocks, and 4-µm sections from each block were used for intraoperative frozen section analysis. Additional sections were stained with HE and the cytokeratinAE1/AE3 (Agilent Technologies) stain for a final postoperative diagnosis.
Occult metastases were classified into 3 categories (8 (link)): Isolated tumour cells (ITCs; <0.2 mm in diameter), micrometastases (0.2-2 mm), and macrometastases (>2 mm). Macrometastasis and micrometastasis were considered indicative of pathological positivity for LN metastasis but ITCs were not.

Yokoyama J., Hasegawa Y., Sugasawa M., Shiotani A., Murakami Y., Ohba S, & Kohno N. (2020). Long term-follow-up multicenter feasibility study of ICG fluorescence-navigated sentinel node biopsy in oral cancer. Molecular and Clinical Oncology, 13(4), 41.

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Formalin-Fixed Paraffin-Embedded Organoid Immunohistochemistry

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Following BME-2 dissociation, organoids were gently pelleted and fixed in 10% neutral formalin before paraffin embedding and sectioning. Paraffin embedded sections of 3.5 μm were stained by a Bond Max autostainer according to the manufacturer’s instruction (Leica Microsystems). Primary antibodies cytokeratin (AE1/AE3, 1:100, Dako), Ki67 (8D5, 1:400, Cell Signalling Technology), and p53 (D07, 1:50, Leica) were applied with negative controls as previously described^{2 (link)}.

Price S., Bhosle S., Gonçalves E., Li X., McClurg D.P., Barthorpe S., Beck A., Hall C., Lightfoot H., Farrow L., Ansari R., Jackson D.A., Allen L., Roberts K., Beaver C., Francies H.E, & Garnett M.J. (2022). A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens. Scientific Reports, 12, 5571.

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Cytokeratin AE1/AE3 Immunohistochemistry

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Immunohistochemical staining was performed according to conventional protocols. In brief, 4-µm-thick formalin-fixed paraffin embedded tissue sections were processed for deparaffinization, antigen retrieval, and endo-blocking and then incubated with primary antibody cytokeratin AE1/AE3 (1:100, Dako, Cambridgeshire, UK). After washing, the slides were incubated with secondary antibody (K5007, Dako Real Envision/HRP) for 30 minutes at room temperature and were then visualized with DAB (K5007, Dako Real DAB+Chromogen) and counterstained with hematoxylin.

Song B., Kim B., Choi S.H., Song K.Y., Chung Y.G., Lee Y.S, & Park G. (2014). Mesenchymal Stromal Cells Promote Tumor Progression in Fibrosarcoma and Gastric Cancer Cells. Korean Journal of Pathology, 48(3), 217-224.

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Comprehensive Histological Evaluation of Tissues

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Trichrome and periodic acid Schiff (PAS) using standard histologic techniques. Tissue was also stained with antibodies to collagen III (BioGenex, San Ramon, CA; Monoclonal clone HWD1.1; at 1:80 titration), cytokeratin AE1/AE3 (Dako, Carpinteria, CA; prediluted), Cytokeratin CAM 5.2 (BD Biosciences, San Jose, CA; prediluted) cytokeratin cocktail (AE1/AE3;CAM5.2), cytokeratin MNF-116 (Dako; at 1/100), and CD34 (Dako; Monoclonal clone QBEND; at 1:320 titration). IHC was performed with automated stainers similar to methods previously published [1 (link), 26 ].

Farris A.B., Ellis C.L., Rogers T.E., Lawson D., Cohen C, & Rosen S. (2016). Renal Medullary and Cortical Correlates in Fibrosis, Epithelial Mass, Microvascularity, and Microanatomy Using Whole Slide Image Analysis Morphometry. PLoS ONE, 11(8), e0161019.

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Histopathological Examination of Cutaneous Carcinosarcomas

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All of the samples were fixed in 10% formalin, routinely processed and embedded in paraffin wax. Then, 4-mm thick sections were stained with hematoxylin and eosin. Furthermore, sections from each case were subjected to appropriately controlled immunohistochemical reactions employing cytokeratin AE1/AE3 (Dako, Glostrup, Denmark), epithelial membrane antigen (EMA; Dako), p53 protein (Dako), p63 protein (Dako), vimentin (Dako), desmin (Dako), and S-100 protein (Dako). All cutaneous carcinosarcomas specimens were evaluated by two dermatopathologists.

Kwak H.B., Park J., Kim H.U., Nam K.H, & Yun S.K. (2018). Cutaneous Carcinosarcoma: a Clinicopathologic and Immunohistochemical Analysis of 11 Korean Cases. Journal of Korean Medical Science, 34(1), e5.

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Multiplex Immunophenotyping of Tissue Samples

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Cytokeratin (AE1/AE3, Dako, 1:50) was used as an epithelial marker, while Mac387, CD163, CD68 and Ham56 (Dako, 1: 20) were used as macrophage markers, DC-SIGN was used as a dendritic cell marker, Langerin was used as a Langerhans cell marker, CD3 as a T cell marker, and CD20 as a B cell marker. Double immunofluorescence-FISH was performed as for ISH, with additional immunofluorescence detection performed by incubating primary antibodies overnight before the detection of signals with the appropriate fluorescent secondary antibodies. ISH signals were detected using anti-digoxigenin alkaline phosphatase conjugate and HNPP/Fast Red TR mix (Roche). The secondary antibody for immunofluorescence was generally anti-mouse fluorescein thiocyanate (FITC) (Invitrogen). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) in anti-fade mounting medium (Vectashield, Vector Laboratories).

Liu L., Wei Q., Nishiura K., Peng J., Wang H., Midkiff C., Alvarez X., Qin C., Lackner A, & Chen Z. (2016). Spatiotemporal interplay of severe acute respiratory syndrome coronavirus and respiratory mucosal cells drives viral dissemination in rhesus macaques. Mucosal Immunology, 9(4), 1089-1101.

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Immunohistochemical Analysis of Tissue Sections

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Paraffin embedded sections of 3.5 μm were stained by a Bond Max autostainer according to the manufacturer’s instruction (Leica Microsystems). Primary antibodies cytokeratin (AE1/AE3, 1:100, Dako), Vimentin (D21H3, 1:100, Cell Signaling Technology), and p53 (D07, 1:50, Leica) were optimized and applied with negative controls.

Li X., Francies H.E., Secrier M., Perner J., Miremadi A., Galeano-Dalmau N., Barendt W.J., Letchford L., Leyden G.M., Goffin E.K., Barthorpe A., Lightfoot H., Chen E., Gilbert J., Noorani A., Devonshire G., Bower L., Grantham A., MacRae S., Grehan N., Wedge D.C., Fitzgerald R.C, & Garnett M.J. (2018). Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics. Nature Communications, 9, 2983.

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Lymph Node Aspiration and Lung Tumor Biopsy Analysis

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The aspiration material obtained from the lymph node was separated into two parts; its small portion was available for cytological investigation. The specimen was air-dried and Hemacolor-stained (MERCK, Darmstadt, Germany) at bedside according to the manufacturer's instructions and a portion was fixed in 95% ethanol and stained with Papanicolaou as ordinary methods. The larger part of the aspiration material obtained from the lymph node and the biopsy material taken from the lung tumor were immediately immersed in 20% buffered neutral formalin, fixed overnight, and embedded in paraffin. These specimens were then sectioned and used for hematoxylin-eosin staining, immunohistochemistry with antibodies for EMA (DAKO, Glostrup, Denmark), p63 (Santa Cruz Biotechnology, Dallas, TX, USA), cytokeratin AE1/AE3 (DAKO), cytokeratin CAM 5.2 (Becton, Dickinson and Company, CA, USA), CD138 (DAKO), vimentin (DAKO), and others, and FISH, as reported previously [8 (link)].

Kuroda S., Suzuki S., Kurita A., Muraki M., Aoshima Y., Tanioka F, & Sugimura H. (2015). Cytological Features of a Variant NUT Midline Carcinoma of the Lung Harboring the NSD3-NUT Fusion Gene: A Case Report and Literature Review. Case Reports in Pathology, 2015, 572951.

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Comprehensive Immunohistochemistry Protocol

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For immunohistochemistry, 2 μm thick sections were cut and antigens retrieved in an epitope retrieval solution of pH 8 (RE7116; Novocastra, Newcastle, UK) at 68 °C for 17 h in a stirred water bath. The antibody clones, dilutions and sources are as follows: CD99 (12E7, 1:25; Dako, Ely, UK), vimentin (V9, 1:100, Novocastra), CD31 (JC70, 1:100, Dako), CD34 (QBend10, 1:50, Dako), cytokeratin AE1/AE3 (1:100, Dako), CD45 (2B11+PD7/26, Dako), cytokeratin MNF116 (1:50, Dako), desmin (D33, 1:100, Dako), α-smooth muscle actin (SMA) (1A4, 1:200, Dako), epithelial membrane antigen (EMA) (E29, 1:100, Dako), HMB-45 (1:200, Dako), S100 (NCL-L-S100p, 1:1000, Novocastra), WT1 (C-19, 1:500, Santa Cruz, Insight Biotechnology Limited, PO Box 520, Wembley, Middlesex,UK), TLE1 (M-101, 1:50, Santa Cruz), ERG (Erg-1/2/3 C-1; 1:50, Santa Cruz), INI1 (1:25, BD Transduction Laboratories (BD Biosciences), Becton Dickinson UK, Oxford, UK), BCOR (C-10, 1:50, Santa Cruz), ETV4 (16, 1:50, Santa Cruz) and Ki-67 (MIB1, 1:200, Dako).

Alholle A., Karanian M., Brini A.T., Morris M.R., Kannappan V., Niada S., Niblett A., Ranchère-Vince D., Pissaloux D., Delfour C., Gonzalez A.M., Antonescu C.R., Sumathi V., Tirode F, & Latif F. (2018). Genetic analyses of undifferentiated small round cell sarcoma identifies a novel sarcoma subtype with a recurrent CRTC1-SS18 gene fusion. The Journal of pathology, 245(2), 186-196.

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