Dual immunohistochemistry is a better immunohistochemical method in scientific research. Its principle is an intuitive multi-color localization staining method based on the difference between positive and positive expression regions. The target of the label is two or more cell sites. The main marking locations are as follows:
1. Membrane + slurry type
2. Membrane + karyotype
3. Pulp + karyotype
Do not film + membrane, nuclear + core, so as not to affect the effect and color overlap. Since the color of the HRP and AEC color development systems is different under the spectacles, direct antigen localization is very intuitive.
Double immunohistochemistry steps:
1, slice dewaxed to water
2. Block endogenous peroxidase (198 ml of methanol or water + 2 ml of 30% H2O2) for 20 minutes at room temperature.
3. Wash the pH 7.2 buffer three times.
4. Trypsin digestion at 37 ° C for 30 minutes. (Or according to the instructions: microwave repair in the antigen repair solution for 20 minutes, or electric furnace for 30 minutes, or 3 minutes after the pressure cooker is capped and vented, rapid cooling; or no treatment)
6. Add normal serum at 37 °C for 30 minutes. (may not be used)
7. Wipe off excess serum and add a primary antibody at 37 °C for 30 minutes.
8. Wash the pH 7.2 buffer three times.
9. Add biotinylated secondary antibody at 37 ° C for 30 minutes.
10. Wash the pH 7.2 buffer three times.
11. Add ABC or SP complex at 37 ° C for 45 minutes.
12. Wash the pH 7.2 buffer three times.
13, DAB or (BCIP / NBT purple blue) color for 3 to 10 minutes.
14. Wash the pH 7.2 buffer three times.
15, double standard blocking solution, 10 minutes (can be used)
16. Add normal serum at 37 °C for 30 minutes. (may not be used)
17. Wipe off excess serum and add a second primary antibody at 37 °C for 30 minutes.
18. Wash the pH 7.2 buffer three times.
19. Add biotinylated secondary antibody at 37 ° C for 30 minutes.
20. Add alkaline phosphatase complex at 37 ° C for 30 minutes.
21, AEC color development
22, washing
23, hematoxylin dyed, blue, dehydrated, transparent, sealed
Precautions:
1. The treatment of slides has many double immunohistochemical steps, and the two heat repairs require higher glue on the slides to avoid peeling.
2. Since the color of the two color rendering systems may overlap, it should be easy to put it on the front, and it is easy to put it behind. Unless otherwise stated, it is still the first out of the HRP system, then the AEC system.
3. Double dyeing should be purposeful, and it should not be blindly double-stained to make the results unconvincing.
Double dyeing application:
1. It is used to distinguish the difficult parts of the conventional HE staining. For example, the lymphatic vessels and the vascular lumen in the vasculature cannot be distinguished by the naked eye.
2. The spread of malignant tumors. The infiltration of malignant tumor cells is chronically phagocytized under normal mucosa. If the epithelial and tumor cells are simultaneously labeled, the infiltration can be observed more directly.
3. Distribution of different cells in the same area.
The above is a detailed introduction of “How to do dual immunohistochemistry experiments†by Shanghai Baiwo Biotechnology. If you have any questions about this, you can search for more information about immunohistochemistry experiments at
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