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Shanghai Yiji Industrial Co., Ltd. is mainly engaged in the research, development and sales of molecular biology, immunology and biochemical reagents.
What are the main points and techniques of immunohistochemistry experiments?
1. Fixation: It is best to use 4% paraformaldehyde fixative. For frozen sections, formaldehyde fixation is sometimes better than frozen acetone; but for different groups and antigens, different fixing solutions can be used. Sometimes the commercially available antibody will have a suitable and recommended fixative solution. Please pay attention to the instructions before purchasing. Bouin S fixative: 750ml of saturated picric acid, 250ml of formaldehyde, 50ml of glacial acetic acid. It has strong penetrating power to tissues, good fixation and complete structure. Not suitable for long-term preservation of tissue specimens. PLP solution: namely sodium periodate-lysine-paraformaldehyde, suitable for fixing paraffin sections. It is suitable for carbohydrate-rich tissues and has good preservation of ultrastructure and antigenicity of many antigens.
2. The tissue is dehydrated and transparent: the time should not be too long, otherwise it will be easily broken and incomplete when sliced.
3. Spreading during sectioning: Some tissues are difficult to spread in water after sectioning, then a few drops of ethanol can be added to the water appropriately.
4. Baking slices: 60 ℃ for 30 minutes or 37 ℃ overnight, the temperature is too high or the time is too long, the antigen is easy to be lost.
5. Preservation of wax blocks and slices: best stored at 4 ℃
6. Off-chip problem: Poly-L-Lysine (poly-lysine) is currently the most commonly used anti-off tablet in immunohistochemical staining work, 6ml of poly-lysine solution can be diluted 1:10 into 60ml The working fluid is suitable for anti-strip processing that requires enzyme digestion, microwave, high temperature and high pressure. If not, double-processing (APES and Poly-L-Lysine) slices can be used. In the case where the above two conditions are not feasible, the following methods can be used: slices are immersed in APES 1:50 acetone solution for 3 minutes before dewaxing, and dried to proceed to the next step.
7. Inactivation of endogenous enzymes: HRP system: 3% hydrogen peroxide inactivation; AP system: 3% HAc inactivation.
8. Antigen exposure: For immunohistochemistry experiments on paraffin sections, high-temperature heating must be used for antigen repair, which will help to expose the antigenic determinants, thereby increasing the strength of immunohistochemical staining (see the antibody manual for the best repair solution for different antibodies) . For different tissues, different antigens, and different antibodies, the method used should be different. It can be heat repaired, trypsinized, and neither repaired nor digested. Collagen can also be digested with pepsin.
9. Blocking: When the goat serum is blocked and the non-specific staining is still strong, the blocking time can be extended or blocked with concentrated serum
10. Antibody dilution: follow the principle of "ready-to-use". Antibodies diluted in PBS must be used on the same day.
11. High background: Under suitable conditions such as antibody concentration, reaction time, reaction temperature, etc., if the background is still high, you can use PBS containing 1 ‰ Tween20, especially before washing.
12. Return to blue: After counterstaining with hematoxylin, use alkaline buffer (such as PBS) or Na2HPO4 saturated solution to return to blue.
13. Color rendering: Be sure to observe under the microscope, pay attention to control the background.
14. During the entire operation, the slices must not be dried, otherwise there will be non-specific staining.
Shanghai Yiji Industrial Co., Ltd. is mainly engaged in the research and development and sales of immunology, molecular biology and conventional biochemical reagents.
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