Identification of ovarian cancer cells was carried out according to the WHO classification of tumors of female reproductive organs (2014).25 For our analysis (of our shown example of endometrioid adenocarcinoma), antigen retrieval for immunohistochemistry was carried out as follows: anti‐Vimentin staining: microwave oven; anti‐estrogen receptor staining: Steamer (EDTA, pH 9); anti‐Ki67 staining: pressure cooker; anti‐CK7 staining: enzymatic pretreatment with pronase. Sections were incubated with the primary antibodies for 30 minutes in a Dako Autostainer (DAKO, Santa Clara, CA, USA). As primary antibodies, the following monoclonal mouse antihuman antibodies were used, diluted with antibody diluent (Zytomed Systems, Berlin, Germany): Vimentin (VIM 3B4, 1:300 dilution; Dako/Agilent [Dako, Santa Clara, CA, USA]), estrogen receptor (6F11, 1:50; Leica, Wetzlar, Germany), CK7 (OV‐TL 12/30, 1:200; Dako/Agilent), and Ki67 (MIB‐1, 1:200; Dako/Agilent). For visualization, a Dako REAL Detection System, Alkaline Phosphatase/RED, Rabbit/Mouse (DAKO, Santa Clara, CA, USA) was used, following the manufacturer's instructions. Slides were counterstained using hematoxylin.
Ck7 ov tl 12 30
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The CK7 (OV-TL 12/30) is a laboratory equipment product from Agilent Technologies. It is designed to perform specific functions within a laboratory setting. However, a detailed and unbiased description of its core function cannot be provided while maintaining conciseness without potential extrapolation.
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Lab products found in correlation
Amacr 13h4, by Agilent Technologies (2 mentions)
Ki 67 mib 1, by Agilent Technologies (2 mentions)
Dako autostainer, by Agilent Technologies (1 mentions)
Antibody diluent, by Zytomed Systems (1 mentions)
Nanozoomer s60 digital slide scanner, by Hamamatsu Photonics (1 mentions)
Gata3, by Cell Marque (1 mentions)
Cell conditioning 1 cc1, by Roche (1 mentions)
Ttf 1 8g7g3 1, by Roche (1 mentions)
Sox10 bc34, by Biocare Medical (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Sp8 inverted microscope, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Cd10 56c6, by Leica (1 mentions)
Automated immunostainer, by Roche (1 mentions)
Vimentin vim 3b4, by Agilent Technologies (1 mentions)
Bond 3 automated system, by Leica (1 mentions)
Hmb45, by Agilent Technologies (1 mentions)
Tfe3 mrq37, by Cell Marque (1 mentions)
Ventana discovery system, by Roche (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
8 protocols using ck7 ov tl 12 30
1
Immunohistochemical Profiling of Ovarian Adenocarcinoma
2
Immunohistochemical Analysis of Tumor Samples
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Patient tumor samples and tumors excised from patient-derived models were stained with IHC and Hematoxylin Eosin (HE). Primary antibodies used for IHC were as follows: HER2 (HER2/neu, Agilent Technologies, Glostrup, Denmark), CK7 (OV-TL12/30, Agilent Technologies, Glostrup, Denmark), CK20 (Ks20.8, Agilent Technologies, Glostrup, Denmark), and GATA3 (L50–823, Cell Marque, The Hague, Netherlands). Glass slide samples were digitized with a NanoZoomer S60 digital slide scanner (Hamamatsu Photonics, Hamamatsu, Japan).
3
Comprehensive Immunohistochemical Panel for Tissue Analysis
4
Immunohistochemical Staining Protocol for Tumor Markers
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Immunohistochemical staining was performed using the following antibodies: CK7 (OV-TL 12/30, 1:100, DAKO, Glostrup, Denmark), CA IX (dilution 1:200, mouse monoclonal, Leica), AMACR (13H4, 1:100; DAKO, Glostrup, Denmark) and TFE3 (1:1500, Santa Cruz Biotechnology Inv., Santa Cruz, CA, USA).
Evaluation of the immunohistochemical staining was performed by light microscopy using a 10× objective lens with the selective use of a 20–40× objective lens for confirmation. The interpretation of immunoreactivity was performed in a semiquantitative manner by analyzing the extent of the staining positivity of the tumor cells. Immunostaining of greater than 10% of tumor cells was required for scoring as a positive case. The interpretation score was as follows: 0 or negative ≤10% tumor cell positivity; +1 or weak = 11–25% tumor cell positivity; +2 or moderate = 26–50% tumor cell positivity; and +3 or strong >50% tumor cell positivity [6 (link)]. Cytoplasmic and/or membranous expression of CK7 and AMACR were considered positive. Only distinct membranous staining for CA IX and distinct nuclear staining for TFE3 were considered positive [15 (link)].
Evaluation of the immunohistochemical staining was performed by light microscopy using a 10× objective lens with the selective use of a 20–40× objective lens for confirmation. The interpretation of immunoreactivity was performed in a semiquantitative manner by analyzing the extent of the staining positivity of the tumor cells. Immunostaining of greater than 10% of tumor cells was required for scoring as a positive case. The interpretation score was as follows: 0 or negative ≤10% tumor cell positivity; +1 or weak = 11–25% tumor cell positivity; +2 or moderate = 26–50% tumor cell positivity; and +3 or strong >50% tumor cell positivity [6 (link)]. Cytoplasmic and/or membranous expression of CK7 and AMACR were considered positive. Only distinct membranous staining for CA IX and distinct nuclear staining for TFE3 were considered positive [15 (link)].
5
Immunofluorescence Staining of Fixed Cells
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PFA-fixed cells or explants were incubated with primary antibodies diluted in PBS/1% BSA IgG free/0.05% saponin. After overnight incubation at 4°C, cells were rinsed three times with 0.1% Tween-20 in PBS (PBST), and staining was revealed with an appropriate secondary antibody and/or phalloidin for 1 h, in the dark at room temperature. After three washes in PBST, cells were counterstained with DAPI for 10 min at room temperature. Finally, slides were mounted with Fluorescent Mounting Medium (#S3023, Dako) and stored at 4°C. Confocal microscopy images were obtained with a Leica SP8 inverted microscope equipped with Plan Apo x40/1.3 and x60/1.4 oil objectives and processed with ImageJ Version 1.52i (National Institutes of Health, https://imagej.nih.gov/ij/ ).
The primary antibodies used were: IFITM1 (#60074-1-Ig, Proteintech) 3 μg/ml, IFITM2/3 (#ab109429, Abcam) 1 μg/ml, HLAG (#MA1-19513, Invitrogen) 1 μg/ml, cytokeratin 7 (CK7, OV-TL 12/30, Dako) 1 μg/ml, FLAG-Tag DYKDDDDK (#F1804, Sigma-Aldrich) 1 μg/ml, and integrin-α5 (ITGA5/CD49e, #IM0770, Immunotech) 2 μg/ml. The secondary antibodies were Highly Cross-Adsorbed Goat anti-Mouse or anti-Rabbit IgG (H+L) coupled with Alexa Fluor 488, 555, or 647 (Invitrogen).
The primary antibodies used were: IFITM1 (#60074-1-Ig, Proteintech) 3 μg/ml, IFITM2/3 (#ab109429, Abcam) 1 μg/ml, HLAG (#MA1-19513, Invitrogen) 1 μg/ml, cytokeratin 7 (CK7, OV-TL 12/30, Dako) 1 μg/ml, FLAG-Tag DYKDDDDK (#F1804, Sigma-Aldrich) 1 μg/ml, and integrin-α5 (ITGA5/CD49e, #IM0770, Immunotech) 2 μg/ml. The secondary antibodies were Highly Cross-Adsorbed Goat anti-Mouse or anti-Rabbit IgG (H+L) coupled with Alexa Fluor 488, 555, or 647 (Invitrogen).
6
Immunohistochemical Profiling of Renal Cell Carcinomas
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Specimens, including 26 CCPRCC, 30 CCRCC and 30 PRCC, were fixed in formalin and embedded in paraffin. The 4-µm thick sections were stained with the following panel of markers: CK7 (OV-TL 12/30, 1:200; Dako, Carpinteria, CA, USA); CD10 (56C6, 1:25; Novocastra, Newcastle upon Tyne, UK); AMACR (13H4, ready-to-use; Dako); CA IX (TH22, 1:100; Novocastra, Buffalo Grove, IL, USA); vimentin (Vim 3B4, 1:250); Ki67 (MIB-1, 1:200) (both from Dako, Glostrup, Denmark). Immunoreaction was performed with an automated immunostainer from Ventana (Ventana Medical Systems, Tucson, AZ, USA). The immunohistochemistry results (CK7, C10, AMACR, CA IX and RCC maker) were interpreted as negative, weak (<30% staining), moderate (30–70% staining) and strong (>70% staining). Ki67 positive cells showed stained brownish-yellow granules in the nucleus. According to the literature written by Delahunt et al (28 (link)), the area with the highest fraction of Ki67-stained cells in section was chosen at a X10 objective magnification, then it was examined at X400 objective magnification. Finally, Ki67 labeling index (Ki67 LI) was made through counting 1,000 cancer cells (percentage of nuclei showing positive staining).
7
Comprehensive Immunohistochemical Profiling of FFPE Tissues
8
Immunohistochemical Analysis of Neuroendocrine Markers
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All 13 patients included in this study were analyzed by IHC. The immunohistochemical analysis was performed on paraffin-embedded sections on a DAKO Autostainer. The primary antibodies used in the study included synaptophysin (SP11, dilution 1 : 100; Thermo Fisher Scientific), chromogranin A (DAK-A3, dilution 1 : 100; DAKO), Ki-67 (MIB-1, dilution 1 : 200; DAKO), cluster of differentiation protein 56 (123C3, dilution 1 : 100; DAKO), CK7 (OV-TL12/30, dilution 1 : 400; DAKO), CK19 (RCK 108, dilution 1 : 100, DAKO), and cytokeratin (AE1/AE3, dilution 1 : 100; DAKO). Appropriate positive and negative controls were used for all antibodies tested. For each immunohistochemical procedure, antigen retrieval was performed in a citrate buffer, and detection was amplified with the DAKO EnVision System. Mitoses were counted in at least 50 HPFs (1 HPF = 2 mm2), and the Ki-67 index was defined using the MIB antibody as the percentage of 500-2000 cells counted in areas of the strongest nuclear labeling (“hot spots”) [6 ].
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