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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


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

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

The detection system and the secondary antibody are not compatible

Ensure that the detection system and the secondary antibody are compatible

The primary antibody and the secondary antibody are not compatible

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).

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

Choose another positive section to detect/ Increase the antibody concentration

The primary/ secondary antibody was not stored properly

Replace the antibody

The substrate is invalid

Replace the substrate

Improper pH of buffers

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

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

Possible causes


Inappropriate antigen retrieval

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

Liquid on the section was not cleaned out, resulting in artificially dilution of antibody

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

The section was not placed horizontally, resulting in loss of antibody

Ensure that the section is horizontally placed during incubation

Improper fixation method

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

3. Non-specific staining

Possible causes


Insufficient deparaffinization of Paraffin-sections

Prolong the deparaffinization time

There is endogenous enzymes or biotin

Remove the endogenous enzymes and biotin effectively

Wrong blocking or insufficient blocking

Use proper blocking buffer or prolong the blocking time

The antibody specificity is low.

Use antibody with better specificity

Insufficient washing

Operate washing in accordance with the experiment process strictly

The primary/ secondary antibody concentration may be too high

Use a lower concentration of antibody

DAB incubation time may be too long

Shorten the DAB incubation time

The sections/cells have dried out

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

There is cross-reactivity between the secondary antibody and endogenous proteins

Avoid using the secondary antibody which has the same species as the sample

Antigen translocation

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


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.

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