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When immunohistochemical staining does not produce the expected results, the cause should be systematically searched, and only one possible cause can be excluded each time.

Control/sample without staining
1 Confirm whether you have ignored some reagents that should be added, including primary, secondary, tertiary and substrate.
2 Verify that all reagents are added in the correct order and that they have been incubated for a sufficient period of time.
3 It is important that the label of the control antibody confirms that the correct antibody is used and whether the detection system used matches the primary antibody. For example, if the primary antibody is a rabbit-derived antibody, the secondary antibody must be matched with an anti-rabbit secondary antibody; or the primary antibody is the mouse IgM primary antibody, and the secondary antibody must be a goat/rabbit anti-mouse IgM (not IgG) )Secondary Antibodies.
4 Check the dilution and dilution solution used for the antibody.
5 Check the expiration date and storage conditions of the antibody, especially the antibody labeled with enzyme or fluorescein. Now most of the reagent company's antibodies are required to be stored at 4~8 °C. Avoid repeated freezing and thawing. Avoid using reagents when storing. Place a chamber with a volatile organic solvent to avoid lowering the potency of the antibody.
6 Check the storage conditions of the specimen. It is best to use a specimen with a known positive to make a positive control at the same time.
7 Check the chromogen/substrate solution. The simplest method is to add a drop of the enzyme-labeled antibody to the prepared substrate solution. If the substrate undergoes the expected color change, the substrate factor can be excluded. It should be noted that some substrates should be used up within a certain period of time after being made into working fluid, otherwise it will be invalid.
8 Check that the rinse is compatible with the reagents. The pH of the solution is important. The solution that matches the peroxidase substrate should not contain sodium azide.
9 Check that the counterstain and sealer match the chromogen used.

Weakly positive
If the negative control is not stained and the positive control specimen is weakly positive, in addition to considering the above factors, it should also be considered:
1 The way the specimen is fixed, the improper fixation method or the high temperature at the time of fixation will affect the quantity and quality of the detected antigen.
2 Inappropriate antigen retrieval method, due to the blocking effect of the fixative on the antigen during the production of paraffin sections, it is necessary to use antigen heat repair or enzymatic digestion or two simultaneous antigen double exposure methods. As for which method is used, Should refer to the manufacturer's instructions, combined with the specific circumstances of the specimen.
3 Is the dilution of the antibody too high or the temperature/time of incubation is correct? General reagent manufacturers will give a certain range of use of reagents, but since the user's specimens come from various tissues, the processing is not the same, so the gradient of the primary antibody used should be tested according to the scope of use. The best use concentration.
4 Excessive rinsing fluid is left on the slice, and when the antibody is added to the slice, it is equivalent to artificially further dilution of the antibody.
5 Whether the slices are placed at the level of incubation, otherwise the antibody will be lost.
    If the negative control does not respond, the positive control responds well, and the specimen is weakly positive, which may be due to the fact that the positive control is not the same tissue, or the fixation method is different.

Non-specific staining
1 Whether the endogenous enzymes and biotin are effectively removed. It should be noted that not every tissue needs to perform this step, but for endogenous enzymes or biotin-rich tissues such as liver, kidney, etc., this reason needs to be considered. The method of processing is:
Inactivation of alkaline phosphatase: The most common method is to add levamisole (24mg/m1) to the substrate solution and keep the pH at 7.6~8.2, which can remove most of the endogenous alkaline phosphatase. Acid phosphatase, which interferes with staining, can be inhibited with 50 mmol/L of tartaric acid.
Saturating endogenous biotin: The method of eliminating endogenous biotin is to add avidin in advance to saturate the endogenous biotin so that it no longer has the remaining binding sites. Specifically, the sections were immersed in a 25 ug/ml avidin solution for 15 minutes before staining by ABC method or SP method, and stained after washing with PBS for 15 minutes.
2 Choose whether to use the correct blocked serum. The non-specific background staining elimination method caused by charge adsorption is to incubate the sections with a non-immune serum of a secondary antibody, diluted with PBS to a 3%-10% solution to block the adsorption site. Sometimes other unrelated proteins, such as bovine serum albumin, are also commonly used. In addition, it is extremely important to avoid bleeding and necrotic areas when taking materials. Recently, some domestic laboratories used 5% skim milk powder instead of serum for antigen blocking, and the effect was good.
3 Whether the selected antibody meets the test requirements. Non-specific staining due to antigenic impureness, antigenic determinants similar to the target antigen in the specimen, can only be achieved by using high-purity, high-valency antibodies or monoclonal antibodies against more specific antigenic determinants. solve.
4 Is the concentration of primary antibody used too high?
5 Is the cleaning sufficient? Strict operating procedures should be followed. Because it contains a certain amount of salt in the buffer, it is also beneficial to reduce background coloration. Usually 0.05mol/l Tris-HCl, 0.15mol/l NaCl has been applied to most dyeing methods, and Tween 20 is added to the solution. . Reagent companies generally provide a buffer formulation when specifically labeled.
6 Is the DBA used correctly? The incubation time and preparation method of DAB can produce some background color. When using concentrated DAB kit, please strictly follow the order of dropping indicated in the instruction manual, pay attention to correct the pH value of distilled water to ensure the correctness of the experimental results; powder DAB When dissolved, there are often some insoluble particles that must be removed by filtration, which may otherwise deposit on the sliced ​​tissue, producing a speckled coloration. In addition, the DAB is not properly stored and oxides can be deposited on the slices. Therefore, the DAB should be stored in a dry place in the dark, and it should be used now. Add H2O2 before use. Excessive incubation times can also cause background staining.
7 Whether the specimen has dried up during the staining process, otherwise it will cause non-specific staining at the edge.
8 Check if the secondary antibody cross-reacts with the endogenous tissue protein of the specimen.

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