Immunohistochemistry is the application of the basic principle of immunology - antigen-antibody reaction, that is, the principle of specific binding of antigen to antibody, which is determined by chemical reaction to develop the color of the labeled antibody (fluorescein, enzyme, metal ion, isotope). Organizing intracellular antigens (polypeptides and proteins) for localization, qualitative and quantitative studies, known as immunohistochemistry or immunocytochemistry.
First, immunohistochemical operation
1. Paraffin sections are dewaxed to water.
2. Incubate 3% H 2 O 2 for 5-10 minutes at room temperature to eliminate endogenous peroxidase activity.
3, rinse with distilled water, immerse in PBS for 5 minutes x2 [If antigen retrieval is required, it can be done after this step.
Antigen heat repair
(1) High-pressure heat repair EDTA (pH 8.0) or 0.01 M sodium citrate buffer solution (pH 6.0) was added to boiling water. Cover the stainless steel pressure cooker, but do not lock it. Place the slide on the metal staining rack, slowly pressurize, soak the slide in the buffer for 5 minutes, then lock the lid and the small valve will rise. After 10 minutes, remove the heat source, place it in cool water, and open the lid when the small valve sinks. This method is suitable for antigen retrieval which is difficult to detect or nuclear antigen.
(2) Heat the boiling heat repair electric furnace or water bath to heat the 0.01M sodium citrate buffer solution (pH 6.0) to about 95 °C, and put it into the tissue chip and heat it for 10~15 minutes.
(3) Microwave heat repair In a microwave oven, a 0.01 M sodium citrate buffer solution (pH 6.0) is heated to boiling, and the tissue chip is placed, and the power is turned off, at intervals of 5 to 10 minutes, and repeated 1-2 times. Suitable antigens are: AR, Bax, Bcl-2, C-fos, X-jun, C-kit, C-myc, E-cadherin, Chromogranin A, Cyclin, ER, Heat shock protein, HPV, Ki-67, MDMZ, p53, p34, p16, p15, P-glycoprotein, PKC, PR, PCNA, ras, Rb, Topoismerase II and the like.
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4, 5-10% normal goat serum (diluted with PBS) blocked, in order to reduce non-specific background staining caused by endogenous peroxidase, incubate for 10 minutes at room temperature, decanted serum, do not wash. Add primary antibody working solution, incubate at 37 °C for 1-2 hours or 4 °C overnight.
5, PBS rinse, 5 minutes x 3 times.
6. Add an appropriate amount of biotin-labeled secondary antibody working solution and incubate at 37 °C for 10-30 minutes.
7. Rinse in PBS for 5 minutes x 3 times.
8. Add an appropriate amount of horseradish enzyme or alkaline phosphatase-labeled streptavidin (measured by a chemiluminescence reaction on a specific instrument) working solution, and incubate at 37 ° C for 10-30 minutes.
9. Rinse in PBS for 5 minutes x 3 times.
10, color developer color for 3-15 minutes (DAB or NBT/BCIP)
11. The tap water is fully rinsed, counterstained, dehydrated, transparent, and sealed.
Second, immunohistochemistry (LP method) steps
1. Slices are routinely dewaxed to water. For antigen retrieval, you can do this step
2. Wash the buffer for 3 min/2 times.
3. To reduce non-specific background staining by endogenous peroxidase, incubate the sections in Hydrogen Peroxide Block for 10-15 minutes.
4. Wash the buffer for 5 min/2 times.
5. Add Ultra V Block and incubate for 5 minutes at room temperature to block non-specific background staining.
(Note: Do not incubate for more than 10 minutes, otherwise it will cause a decrease in specific staining. If the primary antibody contains 5 - 10% normal sheep serum, this step can be omitted.)
6. Wash the buffer for 5 min/2 times.
7. Add primary antibody working solution at 37 °C for 1 - 2 hours. (The specific incubation time and temperature are ultimately determined by the tester)
8. Wash the buffer for 5 min/2 times.
9. Add Primary Antibody Enhancer and incubate for 20 minutes at room temperature.
10. Wash the buffer for 5 min/2 times.
11. Add HRP Polymer (enzyme-labeled secondary antibody) dropwise and incubate for 30 minutes at room temperature.
(Note: HRP Polymer is sensitive to light and should be protected from unnecessary light exposure and stored in opaque vials.)
12. Wash the buffer for 5 min/2 times.
13. Add 1-2 drops of DAB Plus Chromogen (or AEC Plus Chromogen) to 1 ml DAB Plus Substrate (or AEC Plus Substrate), mix and add to the sections and incubate for 3 - 15 minutes. (The specific time is determined by the depth of dyeing.)
14. Tap water is fully rinsed, counterstained, dehydrated, transparent, and sealed.
Remarks: Specific immunohistochemical guidance operations, you can go to http:// website for more detailed information and online consultation!
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