Immunohistochemistry was performed on 4-μm sections from formalin-fixed, paraffin-embedded (FFPE) tissue using a Ventana Benchmark XT automated stainer (Ventana Medical Systems, Tucson, AZ). The following antibodies were used: Melan-A (monoclonal mouse anti-human anti-body, A103; DAKO; 1:100), TFE3 (monoclonal mouse anti-human antibody, MRQ37, Cell Marque; prediluted), PAX8 (monoclonal mouse anti-human antibody, MRQ50, Cell Marque; prediluted), cytokeratin AE1/AE3 (monoclonal mouse anti-human antibody, AE1/AE3, Ventana; prediluted), cytokeratin 7 (monoclonal mouse anti-human antibody, OV-TL 12/30, Dako; 1:800), CAM 5.2 (monoclonal mouse anti-human antibody, BD, 1:100), Cathepsin K (monoclonal mouse anti-human antibody, 3F9, Cell marque, prediluted) and HBM45 (monoclonal mouse antihuman antibody, Dako; 1:60). The corresponding positive and negative controls were shown to be adequate.
Ov tl 12 30
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The OV-TL 12/30 is a laboratory equipment product from Agilent Technologies. It is designed to perform specific functions within a laboratory setting, but a detailed and unbiased description cannot be provided while maintaining conciseness.
Automatically generated - may contain errors
Lab products found in correlation
Ae1 ae3, by Agilent Technologies (3 mentions)
Dak cdx2, by Agilent Technologies (3 mentions)
Benchmark xt, by Roche (3 mentions)
Mrq 50, by Cell Marque (2 mentions)
Mrq37, by Cell Marque (2 mentions)
Ventana benchmark xt, by Roche (2 mentions)
Dako autostainer, by Agilent Technologies (2 mentions)
Alcian blue, by Muto Pure Chemicals (2 mentions)
Cdx2 88, by BioGenex (2 mentions)
Rck108, by Agilent Technologies (2 mentions)
Cd34 qbend 10, by Roche (1 mentions)
M3515, by Agilent Technologies (1 mentions)
Df1485, by Agilent Technologies (1 mentions)
Ventana discovery ultra platform, by Roche (1 mentions)
Leica scn400 histology scanner, by Leica Microsystems (1 mentions)
Cleaved form of caspase 3, by Cell Signaling Technology (1 mentions)
Peroxidase and fluorophore conjugated secondary antibodies, by Cytiva (1 mentions)
Alexa fluor conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Mab1637, by Merck Group (1 mentions)
A0564, by Merck Group (1 mentions)
24 protocols using ov tl 12 30
1
Immunohistochemistry Profiling of Tissue Samples
2
Immunohistochemical Staining Protocol
3
Histopathological Analysis of Tumor Samples
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Tissues of the surgically resected tumor, pellets of the established cell line, and nodules from the mice were fixed in 10% neutral-buffered formalin for 24–72 h and embedded in paraffin. One representative 4 μm thick section of each specimen was stained with H&E, and another section was stained with the polyvalent basic dye Alcian blue (pH 2.5, 4085-2, Muto Pure Chemicals Co. Ltd., Tokyo, Japan). Other sections of the specimens were analyzed using immunohistochemistry (IHC). The following antibodies were used for IHC on the representative slides for each specimen: anti-cytokeratin 20 (CK20) (KS20-8, 1:50 dilution; Dako, Glostrup, Denmark), anti-CK7 (OV-TL12/30, 1:500 dilution; Dako, Glostrup, Denmark), anti-CDX2 (DAK-CDX2, prediluted; Dako, Glostrup, Denmark), and anti-SATB2 (A4B10, 1:1000 dilution; Abcam, Cambridge, MA, USA) antibodies. All IHC tests were performed using a Dako autostainer (Dako, CA, USA) according to the manufacturer’s recommendations. After deparaffinization, the tissue sections were stained using the antibodies described above and then counterstained with hematoxylin.
4
Immunohistochemical Analysis of Lung Markers
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TTF-1 and CK7 IHC was carried out on formalin-fixed, paraffin-embedded tissue 4 μm sections using anti-TTF-1 (mouse, 8GTG3/1, 1:200, Dako, Carpinteria, CA, USA) and anti-CK7 (mouse, OV-TL 12/30, 1:1000, Dako). Antibody incubation and detection were carried out at 37°C on a Menarini intelliPATH FLX (A. Menarini Diagnostics, UK) using Menarini's reagent buffer and detection kits unless otherwise noted. Antigen retrieval was carried out in a pressure cooker in citrate buffer pH6 for 2 min at 123°C. Pan-CK, CD44 and vimentin IHC was carried out on formalin-fixed, paraffin-embedded tissue 4 μm sections using anti pan-cytokeratin antibody (mouse, M3515, 1:60, ER1 20 min Dako), anti CD44 (mouse, DF1485, 1:100, ER2 10 min, Dako) and anti-Vimentin (mouse, V9, CC1 32 min, Roche). Pan-CK and CD44 staining was carried out on the LEICA Bond Max Platform and Vimentin staining on the Ventana Discovery Ultra platform (Roche). Appropriate positive, negative and isotype controls were included with the study sections (not shown). Digital images of whole-tissue sections acquired using a Leica SCN400 histology scanner (Leica Microsystems).
5
Histopathological Analysis of Tumor Samples
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Tissues of the surgically resected tumor, pellets of the established cell line, and nodules from the mice were fixed in 10% neutral-buffered formalin for 24–72 h and embedded in paraffin. One representative 4 μm thick section of each specimen was stained with H&E, and another section was stained with the polyvalent basic dye Alcian blue (pH 2.5, 4085-2, Muto Pure Chemicals Co. Ltd., Tokyo, Japan). Other sections of the specimens were analyzed using immunohistochemistry (IHC). The following antibodies were used for IHC on the representative slides for each specimen: anti-cytokeratin 20 (CK20) (KS20-8, 1:50 dilution; Dako, Glostrup, Denmark), anti-CK7 (OV-TL12/30, 1:500 dilution; Dako, Glostrup, Denmark), anti-CDX2 (DAK-CDX2, prediluted; Dako, Glostrup, Denmark), and anti-SATB2 (A4B10, 1:1000 dilution; Abcam, Cambridge, MA, USA) antibodies. All IHC tests were performed using a Dako autostainer (Dako, CA, USA) according to the manufacturer’s recommendations. After deparaffinization, the tissue sections were stained using the antibodies described above and then counterstained with hematoxylin.
6
Comprehensive Lung Carcinoma Profiling
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This 9-year study from 2009 to 2017 included all cases diagnosed as lung carcinoma, whether biopsied from lung or metastatic sites. Histopathology of all cases and IHC, wherever done, were reviewed. The IHC markers were chosen based on histopathological findings, from a panel comprising CK7 (Leica, clone OV-TL 12/30, prediluted), CK20 (Dako, clone Ks20.8, prediluted), CK5/6 (Dako, clone D5/16 B4, prediluted), p63 (Biogenex, clone 4A4, prediluted), TTF-1 (Biogenex, clone 8G7G3, prediluted), napsin A (Leica, clone IP64, dilution 1:400), synaptophysin (Biogenex, clone Snp88, prediluted), chromogranin A (Dako, clone DAK-A3), neuron-specific enolase (NSE, Biogenex, clone MIG-N3, prediluted), CD56 (Leica, clone 1B6, prediluted), and CDX2 (Biogenex, clone CDX2-88, prediluted). IHC was performed using the Novolink HRP-linked Polymer Detection System with DAB chromogen from Leica Biosystems.
7
Polyclonal Antibody Generation and Immunohistochemical Staining
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A rabbit anti-CADM1 polyclonal antibody directed against the C-terminal 15-amino acid peptide was generated in our laboratory as described previously [24 (link)]. Other primary antibodies used targeted cytokeratin 7 (CK7) (mouse monoclonal OV-TL 12/30; Dako, Glostrup, Denmark), single-stranded DNA (ssDNA) (rabbit IgG; Immuno-Biological Laboratories Co., Ltd., Gunma, Japan), thyroid transcription factor one (TTF1) (mouse monoclonal; Leica Biosystems, Nussloch, Germany), surfactant protein A (SP-A) (mouse monoclonal; Leica Biosystems), a cleaved form of caspase-3 (rabbit polyclonal; Cell Signaling Technology, Danvers, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (Merck Millipore, Billerica, MA, USA). Peroxidase- and fluorophore-conjugated secondary antibodies were obtained from Amersham (Buckinghamshire, England) and Jackson ImmunoResearch (West Grove, PA, USA), respectively.
8
Automated IHC Protocol for Cytokeratins
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Immunohistochemical (IHC) staining was performed on the BenchMark XT (Ventana) automated slide stainer for cytokeratins 5/6 (clone EP24, EP67 (abcam), diluted 1:100) and cytokeratin 7 (clone OV-TL12/30 (Dako), diluted 1:1000) according to the manufacturer’s instructions.
9
Immunostaining of Mammalian Pancreas
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Samples – both whole mount rodent pancreas and 1 mm-thick human specimens - were incubated for 2 days at room temperature with primary antibody (1:100 dilution) in PBST, washed 3 times with PBST, and incubated for 2 days at room temperature with AlexaFluor-conjugated secondary antibody (Jackson, 1:100 dilution) in PBST. For immunostaining of samples derived from frozen clinical specimens, an identical procedure was applied except preceded by overnight blocking in 3% Normal Donkey Serum + 0.3% Triton-X in PBS at room temperature.
List of labeling reagents used in this study:
List of labeling reagents used in this study:
anti-Parvalbumin (rabbit, Abcam ab11427)
anti-Insulin (guinea pig, Dako A0564)
anti-Tuj1 (mouse, Millipore MAB1637)
anti-Col4 (rabbit, Millipore MAB8201)
anti-GFP 488 (rabbit, Thermo-Fisher A21311)
anti-Glucagon (guinea pig, Takara M182)
anti-CD31 (rabbit, Abcam ab28364)
anti-Laminin (rabbit, Millipore AB2034)
anti-Somatostatin (rabbit, Peninsula T-4103)
anti-melanA (mouse, Dako A103)
anti-CK7 (mouse, Dako OV-TL 12/30)
anti-CK20 (mouse, Dako Ks20.8)
Propidium Iodide (Cell Signaling Technologies 4087 S)
DyLight-488 Lectin (Vector DL-1174)
10
Immunohistochemical Analysis of Squamous Cell Carcinoma
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Among the 111 cases, immunohistochemical studies were available for 105 cases (94.6%); 61 peripheral SqCCs (61 out of 63, 96.8%) and 44 central SqCCs (44 out of 48, 91.7%). Six cases were excluded due to the absence of preexisting informed consent for the further use of their human-derived materials. Immunohistochemical staining was performed with antibodies of TTF-1 (1:200, clone SPT24, Novo, Newcastle upon Tyne, UK) and cytokeratin-7 (CK7; 1:50, clone OV-TL 12/30, Dako, Glostrup, Denmark). TTF-1 analysis was integrated as part of evaluating glandular component of SqCCs, as previously described. Each glandular morphology by H&E staining was confirmed by TTF-1 positivity. Representative sections of each SqCC were stained for CK7, which were interpreted and categorized as negative (0%), focally positive (+1, <10%; +2, 10%–50%), and positive (+3, ≥ 50%). For SqCC cases containing a focal glandular (adenocarcinoma) component, CK7 positivity was evaluated only in the SqCC component.
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