Immunohistochemistry Troubleshooting Tips

Immunohistochemistry (IHC) is a technology applying the antigen-antibody specific binding principle to determine the position, qualitative and quantitative properties of intracellular antigen (polypeptide and protein) through the colorimetric chemical reaction of the labelled antibody color reagent (fluorescein, enzyme, metal ion, isotope). When the IHC staining fails, the causes should be systematically analyzed, and only one possible factor can be excluded at each time.

Here Elabscience lists the common Immunohistochemistry troubleshooting.

1. No staining of sample

Possible causes 1: Some reagent or procedure has been ignored, such as primary antibody, secondary antibody, substrate, addition order, incubation time, etc.

Suggestions 1: Record the experiment procedures to ensure that no operation or reagent is forgotten

Possible causes 2: The detection system and the secondary antibody are not compatible

Suggestions 2: Ensure that the detection system and the secondary antibody are compatible

Possible causes 3: The primary antibody and the secondary antibody are not compatible

Suggestions 3: Use a secondary antibody that was raised against the species in which the primary antibody was raised (e.g. primary is raised in rabbit, use anti-rabbit secondary). Be sure that the isotypes of the primary and secondary are compatible (e.g. IgM vs IgG).

Possible causes 4: The target protein is not present or low expressed in the tissue/ The antibody concentration may be too low

Suggestions 4: Choose another positive section to detect/ Increase the antibody concentration

Possible causes 5: The primary/ secondary antibody was not stored properly

Suggestions 5: Replace the antibody

Possible causes 6: The substrate is invalid

Suggestions 6: Replace the substrate

Possible causes 7: Improper pH of buffers

Suggestions 7: Ensure that the pH of buffers applied are in accordance with the experiment requirements

1. Weakly positive (In addition to the same reasons mentioned above, there are also the following situations)

Possible causes 1: Inappropriate antigen retrieval

Suggestions 1: The Paraffin-sections must be treated with heat-induced method (heat-induced epitope retrieval; HIER) or enzymatic digestion or both the two methods at the same time to make the antigen epitope exposed

Possible causes 2: Liquid on the section was not cleaned out, resulting in artificially dilution of antibody

Suggestions 2: Ensure that there is no liquid on the section before adding of antibody

Possible causes 3: The section was not placed horizontally, resulting in loss of antibody

Suggestions 3: Ensure that the section is horizontally placed during incubation

Possible causes 4: Improper fixation method

Suggestions 4: Choose a proper fixation method, ensure the quantity and quality of antigen

1. Non-specific staining

Possible causes 1: Insufficient deparaffinization of Paraffin-sections

Suggestions 1: Prolong the deparaffinization time

Possible causes 2: There is endogenous enzymes or biotin

Suggestions 2: Remove the endogenous enzymes and biotin effectively

Possible causes 3: Wrong blocking or insufficient blocking

Suggestions 3: Use proper blocking buffer or prolong the blocking time

Possible causes 4: The antibody specificity is low.

Suggestions 4: Use antibody with better specificity

Possible causes 5: Insufficient washing

Suggestions 5: Operate washing in accordance with the experiment process strictly

Possible causes 6: The primary/ secondary antibody concentration may be too high

Suggestions 6: Use a lower concentration of antibody

Possible causes 7: DAB incubation time may be too long

Suggestions 7: Shorten the DAB incubation time

Possible causes 8: The sections/cells have dried out

Suggestions 8: Keep sections/cells at high humidity and do not let them dry out

Possible causes 9: There is cross-reactivity between the secondary antibody and endogenous proteins

Suggestions 9: Avoid using the secondary antibody which has the same species as the sample

Possible causes 10: Antigen translocation

Suggestions 10: Refer to relevant literatures to make sure that whether the antigen translocation is caused by the specific treatment of sample

Summary

The method and operation of IHC are not so complicated, but it is not easy to produce high quality staining results. Only to know well the principle and purpose of each operation step of IHC, we can optimize and correct the wrong operations during the experiment.

Positive and negative controls are necessary for IHC. Section which express the target antigen is usually used as positive control. Negative control is generally set by using PBS or non-primary antibody to replace the primary antibody, the other steps are the same. Positive control is used to eliminate the mistakes of experiment method and system, and negative control is used to eliminate the non-specific staining except primary antibody.