One, Introduction to immunization group
Immunohic tissue chemistry, also known as immunocyte chemistry, refers to a specific antibody with a color tensile -tagged antibody in order of tissue cells through antigen antibody reactions and tissue chemical reactions, which qualitatively, positioned, and quantitatively determined the corresponding reactions. New technology. It cleverly combines the specificity of immune response and the visibility of tissue chemistry. With the help of microscope (including fluorescent microscope, electron microscope), the appearance and amplification effect of the microscopy, Polyide, enzymes, hormones, pathogens, and receptors).
Second, the basic principle of immunohistochemical technology
Immunization technology is a comprehensive qualitative, positioning, and quantification; research and testing technology that is closely integrated in form, function, and metabolism. While detecting the pathogen in situ, the relationship between the tissue lesions and the pathogen can also be observed, and the type of infected cells can be confirmed, which helps to understand the pathogenesis and pathological processes of the disease.
Immune enzyme formation technology is to connect enzymes to antibodies through covalent bonds to form enzyme -label antibodies, and then use enzymes to specialize in the substrate to generate non -soluble products or particles with a certain electronic density. Or the positioning of the cell surface and various antigen components in the cell under the electron microscope, according to the site of the enzyme label, it can be divided into direct method (one -step method), indirect method (two -step method), bridge joint method (multi -step method), etc. The antibodies used for tags can be a dopolon antibody or specific monoclonal antibody prepared with immunomas, and it is best to be a strong -efficient monoclonal antibody with strong high -efficiency price. The direct method is to mark the enzyme directly on the first antibody. The indirect method is to check the enzyme label on the second antibody to detect the specific antigen substance in the tissue cell. At present, immune enzymes are generally selected.
Third, immunohistochemical steps
1. Slice, grilling slices 60 ° C, 1h;
2. Show dewaxing and resurrection
Trinopine 10min, 100 % ethanol 5min, 95 % ethanol 5min, 90 % ethanol 5min, 85 % ethanol 5min, 80 % ethanol 5min, 75 % ethanol 5min, 60 % ethanol 5min, 50 % ethanol 5min, 30 % ethanol 5min, tap water 1min, hydrogen peroxide 1min;
3, 1 portion of 30 % H2O2 plus 10 parts of distilled water, room temperature 10min, distilled water was washed 3 times each time;
4, microwave repair
Immerse the slices into 0.01m citrate buffer, and heat the maximum firepower (98 ° C-10 ° C) in the microwave to boiling, cool (about 5-10min), repeated twice;
5. Cool the slice naturally to room temperature, wash 3 times PBS, 5min each time;
6, closed, 5 % BSA, room temperature 20min, throwing off excess liquid;
7, plus one resistance, 37 ℃, 1h, or 4 ℃ overnight;
8, PBS washed 3 times, 3min each time; each time;
9, plus two resistance, 37 ℃, 15-30min;
10, PBS washed 3 times, 3min each time; each time;
11, add SABC, 37 ° C, 30min;
12, PBS washed 3 times, 5min each time; each time;
13, 1ml of distilled water drip the coloring agent, mix well;
14. After the DAB coloring agent is configured, drip it in sliced, room temperature, and mirror detection reaction time (about 5min);
15. Rinse the water of the water and over -distilled water;
16, Su Musu dyed 2min for 2min, rinse water;
30 % ethanol 3min, 50 % ethanol 3min, 70 % ethanol 3min, 80 % ethanol 3min, 90 % ethanol 3min, 95 % ethanol 3min, 100 % ethanol 3min, dietitom 20min;
18, gum sealing, mirror inspection.
Fourth, the analysis of common problems of immunohistochemistry
1. Paraffin slices appear on the process of dyeing
1) Baking slices are not enough, or the temperature is insufficient, it can extend the time and increase the temperature of the grill;
2) Use a glass tablet containing polyinine, you can buy or do it by yourself;
3) Some organizations themselves are easy to drop films, such as bone organizations, etc. During operation, do not rush directly to the organization, rush above the organization, and let it flush the organization;
4) During high temperature repair, the temperature of the temperature may also cause.
2, edge effect
1) The edge of the tissue and the slice are not sticked firmly. The edge tissue is loosened and floated in the liquid. Each cleaning is not easy to wash the reagents below the tissue. Solution: prepare high -quality films (APES or polyinine),, and polymeramine, and polymeramine,,,), APES or polyteinerine), and polymeramine, and polymeramine. Cut out the thin tissue slices as possible, not thick in 4 microns. The preliminary treatment of the tissue shall be standardized to avoid choosing tissue with more necrosis;
2) The reagents on the slice are not fully covered with the tissue. The reagents on the edge are easy to dry first. The concentration is higher than that of the central tissue and deeper dyeing. Solution: The reagent should be fully covered with the organization, and it should exceed the edge of the tissue 2mm. When using a combination of strokes, in order to avoid the impact of the oil agent, the painting circle should be 3-4mm from the edge of the tissue.
3. Organize tissue sliced non -specific dyeing
1) The antibody incubation time is too long and the antibody concentration is high to increase the background color. This can be controlled by shortening the one -resistance/two -resistant incubation time and diluted antibodies. This is the most important one;
2) As soon as a polyxolite antibody is prone to non -specific coloring, it is recommended to try a single cloning antibody;
3) Endogenous peroxidase and biomas are highly tissue in the liver, kidneys (tissue with multiple blood cells), and need to reduce the background stain by extending the excessive time and increasing the concentration of extinguishing agents;
4) Non -specific components are combined with antibodies, which need to strengthen the closed effect by extending the time and proper concentration of animal immunocal closure and appropriate increased concentration;
5) DAB incubation time is too long or concentration is too high;
6) PBS is not sufficient, and the results of residual antibodies are enhanced. It is particularly important to immerse in one anti -anti -, two -resistant or SP incubation;
7) Dry films often appear during the dyeing process, which is easy to enhance non -specific coloring.
4. The immunohistochemical dyeing is negative
1) Errors of antibody concentration and quality problems and antibody sources;
2) Inadequate antigen repair, the tissue with formaldehyde fixation must be fully fixed with antigen to open the antigen table to facilitate the combination of the antibody; it is recommended to try the microwave repair 4 times*6min. Someone has done experiments, which is the best time and number of times. If not, it can be repaired at high pressure;
3) The tissue slicing itself is low in antigen;
4) The serum closed time is too long;
5) DAB incubation time is too short;
6) Increased cells, antibodies fail to fully enter the response to the cells;
7) Starting to do immunohistochemistry, I suggest that you must first make a positive photo to eliminate methods such as antibodies.
1. Consider the high concentration of one resistance;
2. Then adjust the DAB incubation time;
3. Also consider whether the serum closed time is too short;
4. Properly increase the number of immersion and extension of the immersion time after the antibody incubation.