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Immunohistochemistry tips

1. Fixation: It is best to use 4% paraformaldehyde fixative. For frozen sections, formaldehyde fixation is sometimes better than frozen acetone; however, different fixatives may be used for different tissues and antigens. Sometimes commercialized antibodies will be recommended and recommended. Please pay attention to the instructions before purchase.
Bouin S fixative: 750ml of saturated picric acid, 250ml of formaldehyde, 50ml of glacial acetic acid, which has strong penetrating power to the tissue, good fixation and complete structure, but due to acidity, it will damage the original and the tissue shrinks obviously. Not suitable for long-term preservation of tissue specimens.
PLP solution: sodium periodate-lysine-paraformaldehyde, suitable for fixing paraffin sections. It is suitable for enriched in carbohydrate tissue and has good antigenic preservation of ultrastructure and many antigens.

2. Tissue dehydration, transparency: time can not be too long, otherwise it is easy to fragment when sliced, not complete.

3. Slices when slicing: Some tissues are difficult to spread in water after slicing, and a few drops of ethanol can be added to the water as appropriate.

4. Baked slices: 60 ° C for 30 minutes or 37 ° C overnight, the temperature is too high or too long, the antigen is easy to lose.

5. Preservation of wax blocks and slices: preferably stored at 4 ° C

6. Debonding problem: Poly-L-Lysine (polylysine) is the most commonly used anti-offset tablet in the current immunohistochemical staining work. 6ml polylysine solution can be diluted to 60ml according to 1:10. The working fluid is suitable for anti-offset treatment requiring enzymatic digestion, microwave, high temperature and high pressure. If not, double processing (APES and Poly-L-Lysine) slices are available. In the case where the above two conditions are not feasible, the following method can be used: the slice is immersed in APES 1:50 acetone solution for 3 minutes before dewaxing, and dried to carry out the next step.

7. Inactivated endogenous enzyme: HRP system: 3% hydrogen peroxide inactivation; AP system: 3% HAc inactivated.

8. Exposure to antigen: For immunohistochemistry of paraffin sections, high-temperature heating of antigenic repair must be used, which will help to expose antigenic determinants, thereby increasing the intensity of immunohistochemical staining (see the antibody specification for the best repair solution for different antibodies). ). For different tissues, different antigens, different antibodies, the method used should be different, heat repair, trypsin digestion, neither repair nor digestion. Collagen can also be digested with pepsin and the like.

9. Closure: when the goat serum is blocked and the non-specific staining is still strong, the closure time may be extended or closed with concentrated serum.

10. Antibody dilution: The principle of “current use” should be followed. The antibody diluted in PBS must be used on the same day.

11. High background: If the background is still high under suitable conditions such as antibody concentration, reaction time, reaction temperature, etc., it can be washed with PBS containing 1 ‰ Tween20, especially before color development.

12. Return to blue: After counterstaining with hematoxylin, it can be returned to blue with an alkaline buffer (such as PBS) or a saturated solution of Na2HPO4.

13. Color development: Be sure to observe under the microscope, pay attention to control the background.

14. During the entire operation, the slices should not be dried, otherwise there will be non-specific staining.


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