Tissue was available for immunohistochemistry in 24 OGCs. Immunohistochemical stains were performed using the avidin–biotin complex technique with no biotin blocking with antibodies for cytokeratin AE1/AE3 (Dako Corporation, Carpinteria, CA, USA: clones AE1/3, Dilution 1:100), CD68 (Dako Corporation, Carpinteria, CA, USA: clones KP-1, Dilution 1:3000), p53 (Dako Corporation, Carpinteria, CA, USA: clones DO-7, no dilution), and Ki-67 (Dako Corporation, Carpinteria, CA, USA: Mib-1 clone, Dilution 1:160). Negative and positive controls were included with each batch of slides tested.
Ki 67
Manufactured by Agilent Technologies
Sourced in United States
Ki-67 is a laboratory reagent used to detect the Ki-67 protein, which is a cellular marker for proliferation. It is commonly used in immunohistochemistry and flow cytometry applications to measure the fraction of cells in active parts of the cell cycle.
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Lab products found in correlation
5 protocols using ki 67
1
Immunohistochemical Analysis of OGCs
2
Liver Histopathology and Proliferation Assay
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The liver tissue collected for histopathological examination was fixed in formalin and embedded in paraffin. From the prepared paraffin tissues, 3-μm thick sections were cut with a microtome. Hematoxylin-eosin-stained slides were scored according to Suzuki (28 (link)) (hepatic sinusoidal congestion degree, cytoplasmic vacuolization degree, and parenchymal cells necrosis degree). The paraffinized sections were prepared for immunohistochemical staining. After deparaffinization, the sections were rehydrated in graded ethanol solutions. Following antigen binding in a pressure cooker containing EDTA/Tris buffer (pH 9.0), endogenous peroxidase activity was blocked by exposure to 20% H2O2 for 15 minutes. A two-hour incubation was used for the primary antibody, Ki67 (Dako Corporation, Carpinteria, CA, USA). Ki67 expression in positively stained cells was determined by considering nuclear reactivities only in the microscopic field (using 40 × magnification) and identifying the areas of positive Ki67 (brown-colored cells). Ki-67 proliferation index values 0%-1% were considered as low proliferation, 2%-5% as medium proliferation, and 6%-7% as high proliferation.
3
Immunohistochemical Analysis of Breast Lesions
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Biopsy and surgical samples were collected and embedded in paraffin for histological and immunohistochemical analyses. Immunohistochemical studies for GCDFP-15 (mouse monoclonal antibody; Dako Denmark A/S, Glostrup, Denmark), CK 18 (mouse monoclonal antibody; Abcam plc, Cambridge, UK), AR (mouse monoclonal antibody; BioGenex, San Ramon, CA, USA), Her-2 (polyclonal antibody; Dako Denmark A/S), p53 (Novocastra; Leica Mycrosystems GmbH, Wetzlar, Germany), Ki-67 (Dako Denmark A/S), ER (mouse monoclonal antibody; BioGenex), PR (mouse monoclonal antibody; BioGenex), P63 (mouse monoclonal antibody; Abcam plc), calponin (rabbit monoclonal antibody; Abcam plc), and CD 117 (rabbit polyclonal; Dako Denmark A/S) were performed on the paraffin sections.
4
Immunohistochemical Analysis of Oxidative Stress and Apoptosis
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The 5–6 μm thick cross-sections prepared from paraffin blocks were placed on polylysine-coated microscopic slides. Routine immunohistochemical staining was performed according to the method of Kuloğlu and Aydin.12 (link) Primary antibodies (rabbit polyclonal anti-malondialdehyde antibody, ab6463; Abcam, Cambridge, UK, and CASP3 rabbit polyclonal immunoglobulin G, ab2302; Abcam) immunoreactivity were studied with avidin–biotin–peroxidase methodology. Ki-67 (monoclonal mouse, anti-human, clone MIB-1 code 7240, Dako Denmark A/S, Glostrup, Denmark) antibody was performed with automatic painting device (SN:712299; Ventana Medical Systems, Inc., Tucson, AZ, USA). Light microscopy and photography were done using an Olympus BX50 light microscopy (Olympus Corporation, Tokyo, Japan). A histoscore was derived from the distribution (0.1: <25%, 0.4: 26%–50%, 0.6: 51%–75%, 0.9: 76%–100%) and intensity (0: no staining, +0.5: very little staining, +1: little staining, +2: medium, +3: very strong) of staining immunoreactivity (histoscore = distribution × intensity).13
5
Quantitative Immunohistochemical Analysis of Cell Markers
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In both studies (in vivo and in vitro), we used primary monoclonal antibodies to markers Ki-67 (1:75; Dako Denmark A/S), AP-1 (1:200; Sigma-Aldrich Co.), type I procollagen (1:100, LifeSpan Biosciences, Seattle, WA, USA), and SIRT6 (1:200, Alexa Fluor 647) and biotinylated anti-mouse immunoglobulins (Dako Denmark A/S) as secondary antibodies to provide the immunohistochemical reaction. Visualization of the reaction was carried out using the EnVision Detection System kit (Dako Denmark A/S).
In both cases, morphometric investigation was carried out by using a system of computer analysis of microscopic images, which includes a microscope Olympus BX46, a digital camera (Olympus Corporation), and a personal computer based on Intel Pentium 5 and software Morphology 5.2 (Videotest, Saint-Petersburg, Russia).
We measured the optical density (in cu) and the area of immunopositive expression, which was calculated as the ratio of the area occupied by immunopositive cells (or nuclei) and the total area of cells (or nuclei) in the field of view (in %).
In both cases, morphometric investigation was carried out by using a system of computer analysis of microscopic images, which includes a microscope Olympus BX46, a digital camera (Olympus Corporation), and a personal computer based on Intel Pentium 5 and software Morphology 5.2 (Videotest, Saint-Petersburg, Russia).
We measured the optical density (in cu) and the area of immunopositive expression, which was calculated as the ratio of the area occupied by immunopositive cells (or nuclei) and the total area of cells (or nuclei) in the field of view (in %).
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