Immunohistochemical non-specific staining and control method

1. The identification of non-specific staining often occurs at the edges of tissues, collagen fibers and plasma exudates, necrotic tissues and poorly fixed tissue centers. Shows diffuse, uniform background staining. It can also be randomly distributed positive reaction product spots, clusters or blocks. Second, the reasons for non-specific staining 1. Ig binds to Fc receptors more often in frozen sections (especially lymphoid hematopoietic tissue, lymphocytes, monocytes, tissue cell surface), mainly reacts with secondary antibodies, because the primary antibody The dilution is relatively large, so the interference of Fc receptors is basically non-existent. Since formalin fixation destroys most of the Fc receptors, paraffin sections are rare. Methods to avoid: (1) block the sections with normal serum of the same race as the second antibody; (2) use antibodies without Fc segments, but the price is expensive. (The V region and the C1 region do not contain the Fc segment and are digested with Pepsin.) 2. The electrostatically adsorbed antibody is a negatively-charged globulin that can be easily combined with positively-charged tissues, such as collagen fibers. Avoidance methods: (1) Dilute the primary antibody as much as possible; (2) Reduce the charge of the antibody, such as 2.5% NaCl, 0.05mTBS instead of PBS; (3) Treat the sections with detergents such as triton-100, Tween-20, etc. 3. The secondary antibody binds to the Ig in the tissue like a sheep anti-rabbit secondary antibody. The secondary antibody can cross-react with the (human) Ig in the specimen. . Avoidance method: Dilute the secondary antibody with the same 5% normal serum as the tissue to be tested. Such as human serum. 4. The combination of residual aldehyde groups and Ig in the tissue. If aldehydes are fixed and cannot be rinsed thoroughly, the residual aldehyde groups can be combined with Ig. Methods to avoid: (1) Rinse thoroughly; (2) Wash with 0.02% fresh potassium boron oxide solution for 10 minutes. 5. The content of endogenous peroxidase erythrocytes and granulocytes is particularly high and can be recognized under the microscope. Do not treat it as a positive result. Methods to avoid: (1) Paraffin sections were treated with 0.3% hydrogen peroxide-methanol solution; (2) Frozen sections were treated with 3% hydrogen peroxide for 5 minutes (99: 1) because most of the enzymes were not destroyed because they were not embedded; (3 ) For bone marrow smears, it is best to use non-peroxidase-labeled antibodies. 6. Endogenous biotin is sealed with 24 mg / ml of avidin for 15 minutes. 7. The cross-reaction PcAb has high sensitivity, but slightly lower specificity, and is prone to non-specific staining. Avoid methods: (1) Dilute the antibody as much as possible; (2) Use McAb as much as possible. Source: Biotechnology Forum

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