Histopathological sections were stained with rabbit anti-iNOS2 (1:1,000; cat. no. ab15323; Abcam) at 4°C overnight using the streptavidin-biotin complex method for periodontal tissue. Samples were incubated with secondary antibody peroxidase-labeled streptavidin-biotin complex (1:1,000; Dako; cat. no. P0447; Agilent Technologies, Inc.) for 30 min at 37°C. Subsequently, the samples were observed using a fluorescence microscope at ×20 magnification (Olympus Bio-fluorescence Microscope BX53; Olympus Corporation). A total of three optical fields were viewed per section. A total of 12 sections were analyzed per sample and per group. Then the value was calculated by Image J software (National Institute of Health).
Peroxidase labeled streptavidin biotin complex
Manufactured by Agilent Technologies
Sourced in Denmark
The Peroxidase-labeled streptavidin-biotin complex is a laboratory reagent used in various immunoassay and detection techniques. It consists of the protein streptavidin, which has a high affinity for the small molecule biotin, and is labeled with the enzyme peroxidase. This complex serves as a detection system in applications where biotinylated molecules, such as antibodies or nucleic acids, are used.
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2 protocols using peroxidase labeled streptavidin biotin complex
1
Immunohistochemical Analysis of iNOS2 in Periodontal Tissue
2
Immunohistochemical Analysis of EGFR Expression
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EGFR was assessed by immunohistochemistry, IHC, using a streptavidin-biotin complex technique as previously described.23 (link),24 (link) Endogenous peroxidase was blocked in 0.3% H2O2 in PBS for 20 min. Then, antigen retrieval was done in 0.05% protease K (Code no. S3020, Dako, Glostrup, Denmark) in PBS for 10 min at room temperature. The tissue sections were preincubated in PBS for 10 min and then the primary mouse monoclonal antibody (clone 31G7, Zymed labs, South San Francisco, CA, USA) directed against the EGF receptor, diluted 1:100, was added and the sections incubated overnight at 4°C. The secondary biotinylated antibody (goat anti-mouse, Dako, Glostrup, Denmark) and the peroxidase-labeled streptavidin-biotin complex (Dako, Glostrup, Denmark), diluted 1:200, were then added and the samples incubated for 30 min at room temperature. All sections were developed in 0.05% diamino benzidine (Sigma, St Louis, MO, USA) for 5 min and counterstained in Harris haematoxylin (Sigma, St Louis, MO, USA). Finally, the sections were dehydrated through graded alcohol to xylene and mounted in organic mounting medium (Pertex®, Histolab, Gothenburg, Sweden).
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