When immunohistochemical staining does not produce the expected results, the cause should be systematically searched for and only one possible cause can be excluded each time.
Control/sample without staining 1 Confirm whether any reagents that should be added, including primary, secondary, tertiary, and substrate, are ignored.
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 When immunohistochemical staining did not produce the expected results, if the negative control was not stained and the positive control specimen was 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 when the immunohistochemical staining does not produce the expected results, the positive control responds well and the specimen is weakly positive, then