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 rabbit/mouse. The unique polymer auxiliary agent for macromolecular detection system can detect the binding primary antibody better.
Diluent for DAB concentrated solution has been provided in this kit to avoid the influence of the different water acidity and alkalinity on the DAB Chromogenic Agent.
1. Dewax and hydrate the paraffin section.
2. Retrieve antigen of the tissue section according to special requirements of applied primary antibody.
3. Incubate with 3% H2O2 for 10 min to eliminate endogenous peroxidase activity.
4. Wash with PBS or TBS, 2 min× 3.
5. Drain the PBS with absorbent paper, then add Reagent 3 (Normal goat serum) to the section. Incubate for 30 min at 37℃.
6. Dry the liquid around the section with absorbent paper, and draw a circle around the tissue with an oily pen. Add primary antibody with proper dilution ratio, incubate at room temperature or 37℃ for 1~2h or at 4℃ overnight (then rewarm at 37℃ for 30 min). Wash with PBS or TBS, 2 min × 3.
7. Add Reagent 1, incubate at room temperature or 37℃ for 20 min. Wash with PBS or TBS, 2 min×3.
8. Add Reagent 2, incubate at room temperature or 37℃ for 20~30 min. Wash with PBS or TBS, 2 min×3.
9. Add 1 drop (approximately 50 μL) of Reagent 4 (DAB Concentrate) into each 1 mL of Reagent 5 (DAB Substrate), mix fully and the mixed reagent is the DAB Working Solution. Prepare fresh solution before use and the prepared solution should be stored in the dark. Fresh prepared DAB Working Solution is valid within 4 hours and the unused solution must be abandoned.
10. Take control of the DAB coloration period, the color of tan or brownish yellow is the positive signal. Avoid of excessive reaction.
11. Wash the section with deionized water terminate the chromogenic reaction, then operate the procedures of counterstaining, dehydrating, transparentizing and sealing.
Store at 2~8℃, shading light. Avoid of freezing. Valid for 12 months. The reagents are valid within 6 months after opening.