Mastering Immunohistochemistry: Troubleshooting Guide To Success

Immunohistochemistry (IHC) stands as a cornerstone technique in the realm of biomedical research and clinical diagnostics, enabling the visualization and characterization of specific proteins within tissue samples. Despite its widespread application, the journey towards obtaining reliable and reproducible IHC results is fraught with challenges. From antigen retrieval and antibody selection to staining optimization and interpretation, every step in the IHC workflow demands meticulous attention to detail. In cases of failed IHC staining, a systematic analysis of potential factors should be conducted, with each possibility being evaluated individually. In this article, we present to you the common issues encountered during IHC and some troubleshooting guides to counter them.

No staining of sample

Possible causes:

1. Ignored reagents or procedures, such as primary antibody, secondary antibody, substrate, addition order, incubation time, etc.

Suggestion: In experimental procedures, which often involve multiple steps and reagents, overlooking or neglecting even one of these components can significantly affect the outcome. Therefore, it is advisable to record the experiment procedures to ensure that no operation or reagent is forgotten.

2. Incompatibility between detection system and secondary antibody.

Suggestion: Compatibility issues can arise if the secondary antibody is not designed to work with the detection system being used. Hence, it is important to ensure that the detection system and the secondary antibody are compatible.

3. Incompatibility between primary and secondary antibodies.

Suggestion: It is advisable to 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). Additionally, ensure that the isotypes of the primary and secondary are compatible (e.g. IgM vs IgG).

4. Low expression of target protein or low antibody concentration.

Suggestion: If the target protein is not present or is expressed at low levels in the tissue being studied, it can result in weak or non-existent signal. Similarly, using a too-low concentration of primary or secondary antibody can lead to insufficient binding and detection. Increasing the antibody concentration or choosing a different section with higher expression levels can help improve detection sensitivity.

5. Improper storage of antibodies.

Suggestion: Antibodies are sensitive molecules that can degrade if not stored properly. Factors such as temperature, pH, and exposure to light can impact antibody stability. Ensuring proper storage conditions, such as storing antibodies at recommended temperatures and avoiding repeated freeze-thaw cycles, can help maintain their integrity and effectiveness. If the antibody was not properly stored, the only choice left is to replace the antibody.

6. Invalid substrate.

Suggestion: The substrate is a crucial component of many detection assays, as it reacts with enzymes to produce a visible signal. If the substrate is expired, contaminated, or otherwise compromised, it can result in unreliable or non-specific staining. Therefore, replacing invalid substrates with fresh and validated ones can help ensure accurate detection and interpretation of results.

7. Improper pH of buffers.

Suggestion: Buffers play a critical role in maintaining the stability and activity of enzymes and antibodies during detection assays. Incorrect pH levels can affect the performance of these molecules, leading to suboptimal results. Ensuring that buffers are prepared and adjusted to the appropriate pH for the experiment's requirements can help maintain optimal conditions for antibody-antigen interactions and signal detection.

Weakly positive

In addition to the same reasons mentioned above, there are also the following situations:

1. Inappropriate antigen retrieval.

Suggestion: Failure to properly retrieve antigens can result in weak or nonspecific staining. To avoid this, 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.

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

Suggestion: If there is residual liquid on the tissue section before adding the antibody, it can lead to dilution of the antibody solution and potentially affect the sensitivity of detection. Therefore, ensure that there is no liquid on the section before adding antibodies.

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

Suggestion: Ensure that the section is horizontally placed during incubation for uniform exposure to the antibody and improves the consistency of staining across the sample.

4. Improper fixation method.

Suggestion: Using an inappropriate fixation method or inadequate fixation can result in poor antigen preservation and compromised antibody binding. Hence, choosing a proper fixation method based on the tissue type and experimental requirements, and ensuring sufficient fixation time and quality, are essential for optimal staining results.

Non-specific staining?

Possible causes:

1. Insufficient deparaffinization of Paraffin-sections.

Suggestion: If the paraffin is not adequately removed from the tissue sections before staining, it can interfere with antibody penetration and binding. Therefore, it is advisable to prolong the deparaffinization time, ensuring complete removal of paraffin and improving antibody accessibility to the tissue.

2. Presence of endogenous enzymes or biotin.

Suggestion: Endogenous enzymes or biotin present in the tissue can lead to non-specific binding and background staining in immunohistochemistry assays. Hence, removing these interfering substances effectively is essential to obtain accurate and specific staining results.

3. Wrong blocking or insufficient blocking.

Suggestion: Using an inappropriate blocking buffer or insufficient blocking time can result in increased background staining and reduced signal intensity. To avoid this, use proper blocking buffer or prolong the blocking time to minimize non-specific binding and improve the specificity of staining.

4. Low antibody specificity.

Suggestion: Antibodies with low specificity may bind non-specifically unintended targets, leading to false-positive staining. Hence, it is advisable to use antibodies with better specificity, such as those validated for the target of interest, can help improve the accuracy and reliability of staining results.

5. Insufficient washing.

Suggestion: In this case, follow a strict washing protocol according to the experiment's requirements can help ensure thorough removal of unbound molecules and improve the specificity of staining.

6. High antibody concentration.

Suggestion: Using a too-high concentration of primary or secondary antibodies can lead to increased background staining and non-specific binding. To avoid this, using a lower concentration of antibodies can help optimize the signal-to-noise ratio and improve the specificity of staining.

7. DAB incubation time may be too long.

Suggestion: Prolonged incubation with DAB can lead to excessive background staining and reduced contrast between the target and background. therefore, shortening the DAB incubation time can help control background staining and improve the clarity of staining.

8. Drying out of sections/cells.

Suggestion: To prevent this, keep sections/cells at high humidity and do not let them dry out.

9. Cross-reactivity between secondary antibody and endogenous proteins.

Suggestion: Cross-reactivity between the secondary antibody and endogenous proteins in the tissue can lead to non-specific staining. Using a secondary antibody raised in a different species from the sample can help minimize cross-reactivity and improve the specificity of staining.

10. Antigen translocation.

Suggestion: Certain treatments or experimental conditions may cause antigen translocation, leading to misinterpretation of staining results. Therefore, consulting relevant literature and experimental protocols can help determine whether antigen translocation is a potential issue and adjust experimental conditions accordingly to minimize its effects.

While the methodology and execution of Immunohistochemistry (IHC) may not be overly intricate, achieving high-quality staining results poses a challenge. A thorough understanding of the principle and objectives of each operational step in IHC is essential for optimizing procedures and rectifying any errors encountered during experimentation. By unraveling the mysteries behind failed staining and suboptimal outcomes, we aim to empower researchers and diagnosticians alike to achieve success in their IHC endeavors.