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2-step plus is a two-step immunohistochemical broad spectrum detection reagent. It polymerizes monovalent Fab fragments of secondary antibody and enzyme, which replaces the secondary antibody and tertiary antibody in traditional method, can directly amplify the binding signal of antibody-antigen. This method not only retains the specific binding ability of antibody with antigen, but also can effectively avoid space steric hindrance caused by excessive polymer molecules. Compared with the traditional SP three-step method, this kit has the characteristics of simple, rapid, high-sensitivity. This system abandons the using of biotin, so it can avoid background staining by endogenous biotin. It can be used in IHC, in which the primary antibody is monoclonal/polyclonal antibody derived from goat. The unique polymer auxiliary agent for macromolecular detection system can detect the binding primary antibody better.
1. Dewax and hydrate the paraffin section.
2. Make thermal repair or digestion treatment to antigen of the tissue section if necessary according to antigen/antibody situation.
3. Incubate with Reagent 2 (3% H2O2) for 10 min to eliminate endogenous peroxidase activity. Wash with PBS or TBS, 2 min× 3.
4. Add block buffer, incubate at room temperature for 30 min. Shake off any excess liquid.
5. Add primary antibody (Goat-IgG) with proper dilution ratio, incubate at 20~37℃ for 1~2h or at 4℃ overnight (then rewarm at 37℃ for 30 min). Wash with PBS or TBS, 2 min× 3. Dry the section with absorbent paper.
6. Add Reagent 1, incubate at room temperature or 37℃ for 20 min. Wash with PBS or TBS, 2 min×3.
7. Color development with DAB.
8. Wash with deionized water to make the section counterstained, dewatered, transparent, then seal the section.
Store at 2~8℃, shading light. Avoid of freezing. Valid for 12 months. The reagents are valid within 6 months after opening.