Four μm tissue sections were deparaffinized and blocked for endogeneous peroxidase activity by immersion in 0.3% H2O2 in methanol for 20 minutes. Antigen retrieval was performed in Tris/EDTA buffer (10 mM/1mM; pH 9.0) for 20 minutes at 100°C. After cooling for 10 minutes and washing in PBS pH 7.4, nonspecific binding sites were blocked with Serum Free Protein Block (Dako, Glostrup, Denmark) for 10 minutes followed by 1 hour incubation with SMAD4 antibody (Santa Cruz technology, Santa Cruz, CA, USA) at room temperature. Antibody binding was visualized using the BrightVision poly-HRP detection system (Immunologic, Duiven, the Netherlands) with 3,3-diamino-benzidine as chromogen. Sections were counterstained using hematoxylin, dehydrated and coverslipped using Pertex. Pancreas cancer tissue sections were used as negative control, showing no SMAD4 staining [28 (link)]. Control slides in which the primary antibody was omitted, were included. All sections were evaluated using a binocular microscope (Olympus BX 51, Zoeterwoude, the Netherlands).
Brightvision poly hrp detection system
Manufactured by Immunologic
Sourced in Netherlands
The BrightVision poly-HRP detection system is a laboratory equipment used for the detection and visualization of target proteins in various immunoassay techniques. It utilizes a polymer-based horseradish peroxidase (HRP) conjugate to amplify the signal, resulting in enhanced sensitivity during the detection process.
Automatically generated - may contain errors
2 protocols using brightvision poly hrp detection system
1
Immunohistochemical Detection of SMAD4
2
Quantitative Assessment of HBcAg in Mouse Liver
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Two sections separated by approximately 200 μm of formalin-fixed and paraffin-embedded mouse liver were deparaffinized and subjected to heat-induced antigen retrieval in Tris-EDTA glucose (TEG) buffer (pH 9). Following blockage of endogenous peroxidase activity, the sections were blocked in 10% normal serum and incubated with rabbit anti-HBcAg (Dako). The primary antibody was detected and amplified using Brightvision Poly-HRP detection system (Immunologic) and visualized with diaminobenzidine as chromogen. Finally, sections are counterstained in hematoxylin, coverslipped, and digitized using a 20× objective. Quantitative assessment of HBcAg immunoreactivity was estimated as total counts of positive cellular profiles per area of the liver sections. Profile counting is done by image analysis using Visiomorph (Visiopharm).
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