Common problems and countermeasures of immunohistochemistry

Immunohistochemistry is the application of antigen and antibody binding to detect the distribution of macromolecules such as peptides and proteins in cells. This method has strong specificity, high sensitivity, rapid development and wide application, and has become an important research method in many disciplines of biology and medicine. There are many problems in immunohistochemistry. Generally speaking, there are mainly non-specific coloring, incorrect coloring sites, false positives, no positives, positive weak five categories and other minor problems. Now explain the above points separately.

1, non-specific coloring

Non-specific coloring is the coloring outside the theoretically colored area, which is distinct from the background. The main reasons for non-specific coloring are:

1) Causes of specimens, high levels of endogenous biotin in liver and kidney, combined with SABC lead to non-specific coloration, high collagen content in skin and lung tissue, and non-specific coloration due to the negative charge of collagen.

2) Edge effects caused by slicing.

3) The antibody concentration is too high.

4) The tissue has a hemorrhagic or necrotic area during the treatment.

5) Insufficient washing.

6) The color developer is oxidized and the color development time is long.

Solution:

1) The primary antibody should be used as a concentration gradient, and the positive strong background should be selected, and the concentration of the signal to the high ratio should be used as the experimental concentration of the antibody. The concentration and time of the secondary antibody are generally unchanged.

2) Stabilize the experimental conditions and follow the instructions carefully.

3) In the experiment, the slice should be kept horizontal to avoid antibody loss, so that the slice makes the slice dry.

4) The area covered by the reagent should be larger than the area of ​​the slice in the experiment.

5) Wash thoroughly, soak it in PBS pre-warmed at 37 °C when the background is very deep

6) The developer is formulated to prevent oxidation, especially the formulation of the developer is used, preferably sterilized. The color development time should be strictly controlled under the microscope.

2, the coloring site is wrong

There are three cases of colored parts.

Case 1: This is a cytosolic antigen, but the results show that the results are colored in the nucleus.

Reasons and solutions:

1) The repair time is too long and the repair conditions are very strict. At this time, the reaction intensity should be reduced.

2) The tissue is allowed to stand in xylene for too long, and the specimen should be replaced at this time.

3) Antibodies contain anti-nuclear antibodies, which is rare.

4) Causes of specimens, inadvertent handling of specimens will result in coloration always occurring in the same place.

Case 2: This is a nuclear antigen but the result is in the cytosol.

Reasons and solutions:

1) Nuclear antigens are not easily exposed and need to be fully exposed by heat repair or prolonged repair time.

2) The protein is translated in the cytoplasm and then transported to other parts, so it is normal to have pulp staining.

Case 3: This is a membrane antigen but the result is a stain in both the cytosol and the membrane.

Reason: Protein is translated in the cytoplasm and transported to other sites. Membrane proteins in the process of transport may be displayed in the cytosol.

3, false positive

If there is no primary antibody, the positive signal is mainly due to the cross of the secondary antibody. The globulin is a superfamily, and the mammalian species are close to each other and easily cross-react. For example, goat anti-rabbit antibodies can react with globulin in rabbit specimens, and secondary anti-goat anti-mouse can also react with globulin in rats and mice, especially in the case of repair.

4, no positive

the reason:

1) Antigen stability problems, due to the short half-life of many proteins is easily destroyed.

2) During the preparation of the specimen, the baking time is too high and the specimen preparation is not standardized.

3) The reagent was added during the experiment, and the experimental operation was not standardized.

Solution:

1) Strict operation should be carried out in the fixation of specimens to ensure timely fixation.

2) When the wax is dipped, the temperature should not be too high, and the time should not be too long. Usually 2 hours × 2 times. The temperature of the baked sheet is generally about 60 ° C for 2 hours.

3) In the experiment, strictly follow the experimental steps. When the indicators are both monoclonal antibody and multi-resistance, the primary antibody and the secondary antibody should be strictly matched.

5, the weak is positive

Reasons and solutions:

1) The concentration of primary antibody is too low, the solution: increase the concentration of primary antibody

2) The incubation time is too short, the solution: follow the instructions carefully

3) Antibody loss, solution: supplemental antibody

4) The color development time is too short, the solution: control the reaction time under the microscope

5) Improper antigen retrieval methods, there are many methods for antigen retrieval, such as: repair, digestion, no need to repair, finding a suitable method is one of the effective methods to solve the positive weakness.

6, other issues and precautions

In the experiment, especially after the repair, the slices were easily peeled off. What is the reason for slice stripping?

A: The reasons for slicing are as follows. 1). The slice has not been subjected to anti-stripping treatment.

2). There is no baking in time after slicing.

3). The repair time is too long.

Source: BOSTER

Related Links:

• Immunohistochemistry
• Immunofluorescence
• Embedding slice