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.
Mrq 50
Manufactured by Cell Marque
Sourced in Germany
The MRQ-50 is a laboratory equipment product offered by Cell Marque. It is a versatile instrument designed for various scientific applications. The core function of the MRQ-50 is to perform automated tissue staining and processing tasks with precision and consistency.
Automatically generated - may contain errors
Lab products found in correlation
Ventana benchmark xt, by Roche (2 mentions)
Ae1 ae3, by Agilent Technologies (2 mentions)
Ov tl 12 30, by Agilent Technologies (2 mentions)
Mrq37, by Cell Marque (2 mentions)
Clone 56 c6, by Leica (1 mentions)
Benchmark automated immunostainer, by Roche (1 mentions)
Bcl 2, by Agilent Technologies (1 mentions)
Western breeze immunodetection kit, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ac 15, by Abcam (1 mentions)
5 protocols using mrq 50
1
Immunohistochemistry Profiling of Tissue Samples
2
Immunohistochemical Profiling of Renal Tumors
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The Vim3 antibody was commercially designed (EZbiolab Inc., Carmel, USA) using the unique C-terminal ending of Vim3 as the target and testes as described before (for detailed information please see patent by the University of Cologne, Brandenstein/Fries, patent number EP 13160876.2-1405) [21 (link)].
Renal tumors were preevaluated using the following antibodies: alpha-methylacyl-coenzyme A-racemase (AMACR, p504S; Leica;1 : 50, Wetzler, Germany), anti-carbonic anhydrase IX (ab 1083 51/EPR4151/2; Abcam; 1 : 1000, Cambridge, UK), CD117 (c-kit; clone 12E7; DAKO; 1 : 400, Frankfurt, Germany), cytokeratin 7 (CK7; clone OV-T2 12,730; DAKO; 1 : 6000, Frankfurt, Germany), epithelial membrane antigen (EMA; clone E29; Cell Marque; 1 : 500, Darmstadt, Germany), GATA-3 (clone 650-[823]; Cell Marque; 1 : 1200, Darmstadt, Germany), CD10 (clone 56-C6; Novo Castra; 1 : 20), INI-1 (MRQ-27; Cell Marque; 1 : 100, Darmstadt, Germany), PAX-8 (MRQ-50; Cell Marque; 1 : 100, Darmstadt, Germany), vimentin (V9; clone SP20; Thermo Fisher Scientific; 1 : 800, Frankfurt, Germany), and isocitrate dehydrogenase 2 (IDH2; GTX 628487–100; GeneTex, 1 : 100, Eching, Germany).
Renal tumors were preevaluated using the following antibodies: alpha-methylacyl-coenzyme A-racemase (AMACR, p504S; Leica;1 : 50, Wetzler, Germany), anti-carbonic anhydrase IX (ab 1083 51/EPR4151/2; Abcam; 1 : 1000, Cambridge, UK), CD117 (c-kit; clone 12E7; DAKO; 1 : 400, Frankfurt, Germany), cytokeratin 7 (CK7; clone OV-T2 12,730; DAKO; 1 : 6000, Frankfurt, Germany), epithelial membrane antigen (EMA; clone E29; Cell Marque; 1 : 500, Darmstadt, Germany), GATA-3 (clone 650-[823]; Cell Marque; 1 : 1200, Darmstadt, Germany), CD10 (clone 56-C6; Novo Castra; 1 : 20), INI-1 (MRQ-27; Cell Marque; 1 : 100, Darmstadt, Germany), PAX-8 (MRQ-50; Cell Marque; 1 : 100, Darmstadt, Germany), vimentin (V9; clone SP20; Thermo Fisher Scientific; 1 : 800, Frankfurt, Germany), and isocitrate dehydrogenase 2 (IDH2; GTX 628487–100; GeneTex, 1 : 100, Eching, Germany).
3
Immunohistochemical Analysis of Pax8 and Inhibin
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The primary antibodies used were anti-human Pax8 (MRQ-50, prediluted, Cell Marque) and anti-human inhibin (alpha, R1, prediluted, Cell Marque). Immunohistochemical staining for Pax8, and Inhibin was performed on formalin-fixed, paraffin-embedded, 4-µm tissue sections using a Ventana Benchmark automated immunostainer (Ventana, Tucson, AZ, USA). Tissue sections were deparaffinized, and localization of the antigen–antibody complex was achieved using the OptiView DAB detection kit. Antigen retrieval was performed using heat-induced epitope retrieval for 32 min for Pax8, or 64 min for inhibin, in cell conditioning 1 buffer.
Pax8 immunoreactivity was interpreted using nuclear staining alone, whereas inhibin immunoreactivity was interpreted using cytoplasmic staining [2 (link), 10 (link), 11 (link), 19 (link)]. The percentage of Pax8- or inhibin-immunoreactive tumor cells was scored as a proportion. Representative fields were selected and imaged at 400 × magnification using the same microscope. A 5% immunoreactivity cutoff was used to classify a case as positive.
Pax8 immunoreactivity was interpreted using nuclear staining alone, whereas inhibin immunoreactivity was interpreted using cytoplasmic staining [2 (link), 10 (link), 11 (link), 19 (link)]. The percentage of Pax8- or inhibin-immunoreactive tumor cells was scored as a proportion. Representative fields were selected and imaged at 400 × magnification using the same microscope. A 5% immunoreactivity cutoff was used to classify a case as positive.
4
Immunohistochemistry Profiling of Tissue Samples
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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.
5
Protein Expression Analysis of A172 Cells
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A172 protein lysates were prepared in the presence of protease inhibitors, and 100 μg protein were separated on NuPAGE 4-12% Bis-Tris Gels (Life Technologies, Carlsbad, CA). Blots were probed with primary antibodies raised against PAX8 (MRQ-50, Cell Marque), Bcl-2 (Clone 124, Dako), p53 (1C12, Cell Signaling Technology, Beverly, MA), WT1 (6FH2, Dako, Glostrup, Denmark) and β-actin (AC-15, Abcam, UK) according to the manufacturers’ instruction, or that optimised in the current study (1:200 dilution for WT1). Alkaline phosphatase conjugated antibodies were detected using the Western Breeze Immunodetection kit (Life Technologies, Carlsbad, CA).
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