Back to your search result

Description

Intended Use The IHC Select Immunoperoxidase Secondary Detection System is intended for use with the CHEMICON IHC Select Prediluted Primary Antibody reagents, which contain rabbit and mouse IgG for the qualitative identification of antigens by light microscopy in paraffin-embedded tissues. The IHC Select Immunoperoxidase Secondary Detection System may also be used with other rabbit or mouse primary antibodies. General Purpose Reagents Principle of Procedure: The IHC Select Immunoperoxidase Secondary Detection System uses biotin-avidin-HRP complexed antibodies to detect rabbit or mouse IgG in the Primary Antibody. The primary antibody binds to specific antigens present in the specimen. Any excess antibody is removed by washing. Next, the Secondary Antibodies Reagent (conjugated to biotin) is added and will react with the primary antibody present on the tissue. Unbound antibody is removed by washing. The sample is then by incubated with the Streptavidin-HRP solution, which binds to the biotin-labeled secondary antibody present on the tissue. Unbound enzyme is removed by washing. The chromogenic development reagent, 3,3&prime, diaminobenzidine (DAB Substrate), is then added and reacts with the HRP attached to the HRP-streptavidin-biotin-antibody complex. The HRP activity on the chromogenic substrate results in the deposit of brown to black insoluble precipitate at those antigenic sites containing the specific epitopes recognized by the primary antibody. Tissues processed in a variety of fixatives including neutral buffered formalin, B-5, and Bouin&prime,s may be used. Summary and Explanation: Immunohistochemical techniques have been in use for the recognition of cells in tissue sections for at least 50 years. The first method reported use of fluorescent labels in 19411. In 1974, reports of using enzymatic labels, such as horseradish peroxidase (HRP) in routine H & E paraffin sections appeared2,3. The methods have since become the &prime,standard of care&prime, in surgical pathology when classic methods alone fail to yield a definitive diagnosis4-9. Early techniques were based on the peroxidase-anti-peroxidase (PAP) reaction, while improvements exploited the strong affinity of avidin for biotin10. This technique uses HRP-labeled streptavidin. The secondary antibody is conjugated with biotin and the Strepavidin-HRP complex reacts with the biotin on the secondary antibodies. The resulting biotin-avidin-HRP complex can react with the primary antibody bound to the specific epitope of the target antigen. The HRP enzymes of the streptavidin complex then catalyze the substrate/chromogen reaction to form a coloured reaction product (brown to black when using DAB as the chromogen) at the antigen site. A biological stain (counterstain) is then used to visualize the tissue structure.