EliVisionTM plus/HRP Immunohistochemistry Staining Procedure
Ready-to-use Non-Biotin Immunohistochemistry EliVisionTMplus/HRP Detection Kit is based on polymer technology that conjugates multiple anti-mouse and anti-rabbit IgG molecules with horseradish peroxidase onto a polymer. The formed polymer binds to the primary antibody, thereby amplifying the signal of antigen-antibody binding. The staining procedure is as follows:
1. Deparaffinize and hydrate paraffin sections, rinse with tap water;
2. Perform appropriate antigen retrieval on the tissue according to the requirements of each antibody;
3. If necessary (if the tissue contains endogenous peroxidase), apply peroxidase blocking reagent to the sections, incubate at room temperature for 10 minutes, rinse with PBS three times, each for three minutes (3 × 3 minutes);
4. Remove PBS solution, apply the primary antibody to the sections, incubate at room temperature for 60 minutes or at 4°C overnight, rinse with PBS 3 × 3 minutes;
5. Remove PBS solution, apply reaction enhancer to the sections, incubate at room temperature for 20 minutes, rinse with PBS 3 × 3 minutes;
6. Remove PBS solution, apply enzyme-labeled anti-mouse/rabbit IgG polymer to the sections, incubate at room temperature for 30 minutes, rinse with PBS 3 × 3 minutes;
7. Remove PBS solution, apply freshly prepared DAB or AEC chromogenic reagent to the sections for color development;
8. Rinse with tap water to stop color development, counterstain with hematoxylin (if necessary, differentiate with 1% hydrochloric acid ethanol), return to blue with PBS. If DAB is used for color development, dehydrate the sections through a graded ethanol series, clear with xylene, and mount with neutral resin. If AEC is used for color development, the sections cannot be dehydrated with ethanol and should be directly mounted with an aqueous mounting medium.
1. Deparaffinize and hydrate paraffin sections, rinse with tap water;
2. Perform appropriate antigen retrieval on the tissue according to the requirements of each antibody;
3. If necessary (if the tissue contains endogenous peroxidase), apply peroxidase blocking reagent to the sections, incubate at room temperature for 10 minutes, rinse with PBS three times, each for three minutes (3 × 3 minutes);
4. Remove PBS solution, apply the primary antibody to the sections, incubate at room temperature for 60 minutes or at 4°C overnight, rinse with PBS 3 × 3 minutes;
5. Remove PBS solution, apply reaction enhancer to the sections, incubate at room temperature for 20 minutes, rinse with PBS 3 × 3 minutes;
6. Remove PBS solution, apply enzyme-labeled anti-mouse/rabbit IgG polymer to the sections, incubate at room temperature for 30 minutes, rinse with PBS 3 × 3 minutes;
7. Remove PBS solution, apply freshly prepared DAB or AEC chromogenic reagent to the sections for color development;
8. Rinse with tap water to stop color development, counterstain with hematoxylin (if necessary, differentiate with 1% hydrochloric acid ethanol), return to blue with PBS. If DAB is used for color development, dehydrate the sections through a graded ethanol series, clear with xylene, and mount with neutral resin. If AEC is used for color development, the sections cannot be dehydrated with ethanol and should be directly mounted with an aqueous mounting medium.