Immunoperoxidase labeling assay of tissue sections
Immunoperoxidase labeling assay of tissue sections
This is a very sensitive method. The antibody used to form the HRPO-antiperoxidase (PAP) complex must be of the same type as the primary antibody recognized by the bridging antibody.
Operation method
basic program
Materials and Instruments
Tissue Samples Move 1. Wet paper towels are placed on the bottom of the slide box to make a humidified box, and the slides containing the sections are taken out of the cryostat and placed into the slide box (6 slides on each side) or into the humidified box (the slides should not touch each other).2. Allow the slide to reach room temperature before it dries and spread PBS on the section (do not overflow the slide).3. Incubate in PBS containing 0.25% hydrogen peroxide for 30 min at room temperature and wash three times with PBS. For more product details, please visit Aladdin Scientific website.
PBS hydrogen peroxide PAP diaminobenzidine
Centrifuge Humidifying box Incubator
4. Diluted first Hangers were centrifuged at 4°C in a microcentrifuge at 13 500 g for 2 min (40-50 μl of antibody was added to each slide, which should cover the sections).5. Using a Pasteur pipette attached to the pump, aspirate the PBS from the slide at one end of the section and add the antibody from the other end, cover the humidor and incubate for 1 h at room temperature.
6. PBS the slide 3 times (5 min/times), add new PBS buffer from one end of the slide and aspirate the old buffer from the other end.7. The slides were incubated with specific bridging secondary antibody at room temperature for 1 h. The slides were washed three times with PBS.
8. Add PAP complex and incubate at room temperature for 1 h. Wash 3 times with PBS.9. Color development of DAB substrate solution at room temperature for 2~5 min (the length of color development time depends on experimental experience).
10. Sample and fix the slides, and store them in a slide box in a dark place (no need to freeze them).