Immunohistochemical analysis was performed as described before [48 (link)]. Briefly, 4μm thick sections were stained with hematoxylin and eosin, as well as CD34, CD31, laminin, Vimentin, Pax8, RCC, EMA Pan-Cytokeratine and low molecular weight cytokeratins. The primary antibodies anti-CD34, anti-CD31, anti-Pax8 and anti-Vimentin (pre-diluted; Ventana Medical Systems, Tucson, AZ, USA), anti-LMWCK (1:20; Becton Dickinson, Franklin Lakes, NJ, USA), anti-CD44 (NeoMarkers, Fremont, CA, USA) and anti-laminin (1:20; Agilent Technologies, Glostrup, Denmark) were used for staining on a Benchmark staining platform (Ventana Medical Systems), with the BMK iVIEW DAB Paraffin detection kit (Ventana Medical Systems). After drying, histologic images were obtained by ScanScope XT digital scanning system (Aperio Technologies Inc., Vista, CA) at 20x or 40x magnification. CD34 and CD31 were also used to stain mouse liver and lung and confirmed no-cross-reaction with the mouse background (data not shown).
Benchmark staining platform
Manufactured by Roche
Sourced in Denmark
The Benchmark staining platform is a laboratory equipment designed for automated tissue staining. It provides standardized and consistent staining results for immunohistochemistry and in situ hybridization applications.
Automatically generated - may contain errors
2 protocols using benchmark staining platform
1
Immunohistochemical Analysis of Tissue Markers
2
Immunohistochemical Analysis of Fascin-1 and Galectin-3
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All of surgical specimen slides of these 58 patients were stained immunohistochemically at Benchmark staining platform (Ventana Medical Systems Inc) by Ventana Universal DAB detection kit. As primary antibodies, Fascin-1 (clone FCN01, Neomarkers, Fremont, CA, USA; dilution 1:100) and Galectin-3 (clone 9C4, Leica Biosystems, Novocastra, Newcastle Upon Tyne, UK; dilution 1:100) were used. After staining, sections were dehydrated and closed. For each marker, least 1000 cells were counted at light microscope. For Galectin-3; more than 5% staining was considered as positive, %5 and less was accepted as negative. For fascin-1; staining intensity was categorized in 4 groups; 0: no staining; 1: weak staining; 2: moderate staining; 3: strong staining. The percentage of positive cells was grouped from 1 to 4 as follows; 1: ≤ 10 %; 2:10-50 %; 3: 50-75 %; 4: ≥75. Two scores gathered and final score 4 and less accepted as low staining; 5 and more accepted as high staining.
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