When the expected result does not appear in immunohistochemical staining, the cause should be systematically found, and only one possible cause can be ruled out at a time.
The control / specimen is not stained. â‘ Confirm whether a certain reagent should be added, including primary antibody, secondary antibody, tertiary antibody and substrate.
â‘¡ Confirm that all reagents are added in the correct order and that they have been incubated for a sufficient amount of time.
â‘¢ It is very important that the label of the control antibody confirms whether 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 a mouse IgM primary antibody, and the secondary antibody must be goat / rabbit anti-mouse IgM (not IgG) )Secondary Antibodies.
â‘£ Check the dilution and dilution solution used for the antibody.
⑤ Check the expiration date and storage conditions of antibodies, especially those labeled with enzymes or fluorescein. Most antibodies of reagent companies now require storage at 4 ~ 8 ℃. Avoid repeated freezing and thawing. Place it in the same room as the volatile organic solvent to avoid reducing the antibody titer.
â‘¥ Check the storage conditions of specimens. It is best to use specimens with known positives as a positive control.
⑦ Check the chromogen / substrate solution. The simplest detection method is to add a drop of antibody labeled with enzyme to the prepared substrate solution. If the expected color change of the substrate occurs, the substrate factor can be excluded. It should be noted that some substrates should be used up within a certain time after they are made into working fluid, otherwise they will fail.
⑧ Check whether the rinsing solution matches the reaction reagent. The pH value of the solution is very important. The solution matching the peroxidase substrate should not contain sodium azide.
⑨ Check whether the counter-staining agent and the mounting tablet match the chromogen used.
Weak positive If the negative control is not stained and the positive control specimen is weakly positive, in addition to the above factors, you should also consider:
â‘ The fixation method of the specimen, improper fixation method or high temperature during fixation will affect the quantity and quality of the detected antigen.
② Inappropriate antigen repair method, due to the sealing effect of the fixative on the antigen during the production of paraffin sections, it must be used for antigen heat repair or enzyme digestion or two antigen double exposure methods used at the same time, as to which method is used, Reference should be made to the manufacturer ’s instructions, as well as the specific circumstances of the specimen.
â‘¢ Whether the dilution of antibody is too high or the temperature / time of incubation is correct. General reagent manufacturers will give a certain range of reagents, but because the user's specimens come from various organizations, the processing process is also different, so you should refer to the scope of use, perform a gradient test on the primary antibody used to find out The best use concentration.
â‘£ There is too much washing fluid left on the slice. When the antibody is added to the slice, it is equivalent to further artificial dilution of the antibody.
⑤ Whether the slices are placed horizontally during 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, it may be because the positive control is not the same tissue, or the fixing method is different.
Non-specific staining â‘ Whether the endogenous enzymes and biotin have been effectively removed. It should be noted that not every tissue needs this step, but for tissues rich in endogenous enzymes or biotin, such as liver and kidney, this reason needs to be considered. The treatment method is: inactivation of alkaline phosphatase: the most commonly used method is to add levamisole (24mg / m1) to the substrate solution and maintain the pH at 7.6 ~ 8.2, that is, it can remove most of the endogenous alkaline Phosphatase, for acid phosphatase that can still interfere with staining, can be inhibited with 50mmol / L tartaric acid. Saturation treatment of 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 remaining binding sites. The specific method is to immerse the slice in 25ug / ml avidin solution for 15 minutes before staining by ABC method or SP method, and then stain after washing with PBS for 15 minutes.
â‘¡ Whether the correct blocking serum is used. The method of eliminating non-specific background staining caused by charge adsorption is to incubate the sections with non-immune serum of the secondary antibody animal diluted with PBS to a 3% -10% solution to block the adsorption sites. Sometimes other unrelated proteins, such as bovine serum albumin, are also commonly used. In addition, it is also very important to avoid bleeding and necrosis areas when taking materials. Recently, some domestic laboratories have used 5% skimmed milk powder instead of serum for antigen blocking, and the effect is also good.
â‘¢ Whether the selected antibody meets the test requirements. Non-specific staining due to impure antigens, specimens containing epitopes similar to the target antigen, etc. can only be obtained by using high-purity, high-efficiency antibodies or monoclonal antibodies directed against more specific epitopes solve.
â‘£ Is the concentration of primary antibody used too high?
⑤ Whether the cleaning is sufficient. Strict operating procedures should be followed. Because it contains a certain amount of salt in the buffer, it is also helpful to reduce background coloring. Usually 0.05mol / l Tris-HCl and 0.15mol / l NaCl are suitable for most dyeing methods. Tween 20 is added to the solution for better results . For special marking, reagent companies generally provide buffer formulations.
⑥ Is the use of DBA correct? DAB's incubation time and preparation method can produce some background colors. When using the concentrated DAB kit, please strictly follow the dripping sequence indicated in the instructions, pay attention to correct the pH of distilled water to ensure the accuracy of the experimental results; powder DAB When dissolving, there are often some insoluble particles, which must be removed by filtration, otherwise they may be deposited on the sliced ​​tissue, resulting in spotty coloring. In addition, if the DAB is not properly stored, oxides can be deposited on the slices, so the DAB needs to be stored in a dry place protected from light. Use it now, add H2O2 before use. Long incubation time will 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 whether the secondary antibody cross-reacts with the endogenous tissue protein of the specimen.
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