Principles of Immunohistochemistry
Immunohistochemical staining is to use a primary antibody to bind to the target protein antigen in the tested tissue, and then use a secondary antibody labeled with HRP to bind to the primary antibody, and finally react with DAB chromogenic reagent to confirm the location of the protein antigen to be detected. semi-quantitative purposes.
The more significance of immunohistochemistry is to check the localization of the target protein. Quantification is generally achieved by western.
The difference between immunohistochemical staining and HE staining may be that HE staining can roughly identify the morphological changes of different cells; while the latter can detect the changes (number) of specific cells against specific cell markers, and can also detect intracellular changes. The translocation of cytokines, the changes in the expression of some specific proteins in the tissue (the comprehensive analysis of the color depth and distribution area through the image analysis system).
In layman’s terms, HE only looks at gross pathological changes, such as tissue necrosis, such as inflammatory exudation. Immunohistochemistry to see the expression of your target protein.
Comparison of immunohistochemical DAB and HE staining
DAB is also a dye like HE. HE staining is relatively simple and can distinguish acidophilic substances in cells; the full name of DAB staining should be called immunohistochemical DAB staining. After a series of complex biochemical reactions, DAB ( Diaminobenzidine) dye can dye horseradish peroxidase brown.
Commonly used immunohistochemical staining methods:
If HRP is used for histochemistry, the signal is not strong enough. HRP is usually labeled with biotin to enhance the signal.
1. ABC method (avidin-biotin-peroxidase complex method)
The ABC method uses the high affinity characteristic of avidin and biotin to combine with the biotinylated secondary antibody to form a complex of antigen + specific antibody + biotinylated secondary antibody + avidin + biotin + HRP, and finally DAB develops color.
Compound preparation: first combine biotin with enzyme to form biotinylated HRP, and mix biotinylated HRP and avidin in a certain proportion to form avidin-biotin-peroxidase complex.
2. SP method (Streptavidin-peroxidase complex)
It is not linked to biotin itself, but has two binding sites with high affinity for biotin, which can bind to biotin on the secondary antibody, with high sensitivity, and the complex does not need to be mixed before use, which is more convenient.
3. PAP method
4. Direct method
Analysis of immunohistochemical results
Judgment principles of immunohistochemical results:
1. Positive and negative controls must be set.
2. Antigen expression must be at a specific site.
3. Negative results cannot be regarded as non-expression of the antigen.
Several situations and reasons for dyeing failure:
1. All the stained sections were negative, including the positive control.
2. All sections were positive.
3. The background of all slices is too dark.
4. The positive control stained well, and the positive samples tested were negative.
All sections were positive for the following reasons:
1. The antibody is too concentrated during the staining process, or the slices are dry.
2. If sodium chloride is not added in the buffer configuration, the pH value is inaccurate, and the washing is not thorough.
3. Use the discolored color-forming substrate solution, or the color-forming reaction time is too long.
4. Antibody incubation time is too long.
5. The concentration of H2O2 is too high, the coloring speed is too fast and the adhesive is too thick.
The background of all slices is too dark, the reasons are:
1. Endogenous peroxidase is not completely blocked.
2. The slice or smear is too thick.
3. Not enough rinsing.
4. The color reaction of the substrate is too long.
5. Insufficient protein blocking or hemolysis of serum used.
6. The use of whole serum antibody dilution is not enough.
The positive control stained well, and the positive samples tested were negative.
Most common cause: Improper specimen fixation and handling.
1. The time of hematoxylin counterstaining needs to be explored, especially the location of positive staining.
2. The sections should be fully dewaxed and hydrated; the tissue should be fully covered when adding the reaction solution; the washing solution should be dried before each addition, but the slices should be prevented from drying out.
3. The following reasons may cause uneven coloring of the film:
①Insufficient dewaxing. It can be baked at 60°C for 20min and immediately put into fresh xylene.
②Incomplete hydration. Fresh graded ethanol should be prepared frequently.
③ The antibody is not mixed. Mix reagents such as primary/secondary antibodies thoroughly with a pipette. ④ When the antibody is incubated, the section is tilted.
⑤ Insufficient PBS washing after antibody incubation.
⑥ The thickness of the film is uneven and other problems.
⑦ The dyeing box is not flat, and the slices are inclined.
4. Cleaning of the primary antibody:
①Rinse separately to prevent contamination caused by cross-reaction.
②Rinse gently to prevent the slices from falling off. It is recommended to use the dipping method.
③ The rinsing time should be sufficient to thoroughly wash off the bound substances.
5. Use of PH and ionic strength of PBS:
① The recommended pH is 0.01 M at a concentration of 7.4-7.6.
② Neutral and weak alkaline conditions (PH7-8) are conducive to the formation of immune complexes, while acidic conditions are conducive to decomposition; low ionic strength is conducive to the formation of immune complexes, while high ionic strength is conducive to decomposition.
6. Take pictures: If possible, take pictures immediately. If you can’t take pictures in time, you should seal the film and seal it with nail polish to keep it away from light and humidity.