Immunohistochemistry (IHC) Troubleshooting Guide

Common IHC Troubleshooting Issues

The following guide serves as a checklist for identifying and resolving common protocol issues encountered during IHC staining. Addressing these challenges effectively can save time, and resources, and ensure high-quality staining results.

In July 2020, our team interviewed industry experts and composed an in-depth interview for topics of optimizing and troubleshooting IHC. It answers many interesting questions and you can see more details here: Boster Interview Series: Expert Tips on IHC

#1 No Or Weak Staining

Good IHC results rely on strong, specific staining of the target antigen. Weak or lack of staining often requires repeating the experiment, which can be costly in terms of time and resources. Use the table below to identify and correct the source of weak staining.

S.No.	Possible Cause	Solution
1	Slides lose signal over time during storage	Prepare slides with freshly-sliced tissues. Store slides at 4°C. Do not bake slides before storage.
2	The antibody used is not suitable for IHC procedures which detect proteins in its native conformation	Check the antibody datasheet to make certain that it has been validated for IHC applications. Check the antibody is applicable to the right IHC samples (paraffin sections vs. frozen samples). Perform Western blot with both its native and denatured forms to ensure that the antibody detects the native form.
3	Fixation procedures (using formalin and paraformaldehyde fixatives) have masked the epitope that the antibody recognizes	Use different antigen retrieval methods to unmask the epitope (HIER or PIER) Fix the sections in a shorter time
4	The primary and/or secondary antibody has lost its activity due to improper storage, dilution, or excessive freezing and thawing	Run positive controls to ensure that the primary and/or secondary antibody is working properly. Store the antibodies according to manufacturer instructions. Avoid contamination and light on antibodies.
5	Insufficient deparaffinization	Increase the deparaffinization time Use fresh dimethylbenzene
6	The protein is located in the nucleus and the antibody cannot penetrate the nucleus	Add a permeabilizing agent (e.g. Triton X) to the blocking buffer and antibody dilution buffer
7	The PBS buffer has contaminated with bacteria that damage the phosphate groups on the protein of interest	Add 0.01% azide in the PBS antibody storage buffer Use fresh sterile PBS
8	The primary antibody and the secondary antibody are not compatible	Use a secondary antibody that was raised against the species in which the primary was raised (e.g. if the primary antibody was raised in mouse, an anti-mouse secondary antibody should be used) Check that the isotypes of the primary and secondary are compatible
9	The protein is not present in the tissue of interest or has not sufficiently expressed	Run positive controls to ensure that target protein is present in the tissue Include an amplification step in your protocol Use higher antibody concentration
10	Insufficient antibody to detect protein of interest	Use a higher antibody concentration Incubate for a longer time (e.g. overnight) at 4°C
11	Tissues dried out	Cover the tissues in liquid at all times during the experiment.
12	Enzyme/substrate reaction impeded	Deionized water can sometimes contain peroxidase inhibitors. Use Boster antibody diluent buffer to ensure enzymatic activity. Optimize substrate pH to increase reaction intensity.
13	Buffer incompatible with enzyme	Do not use phosphate buffer with AP system. Do not use sodium azide with HRP system.
14	Inadequate antigen retrieval	Reduce the length of fixation step. Use a different antigen retrieval method. Perform antigen retrieval for longer.

For a more detailed exploration of weak staining issues, check out our advanced guide on troubleshooting IHC experiments.

#2 High Background Staining

High background staining can obscure critical tissue features, making accurate analysis difficult. This issue often arises from non-specific antibody binding of primary and/or secondary reagents to tissues or preparation errors. You can typically expect some amount of background staining during IHC. However, once the level of background staining becomes high enough to obscure important features and structures of the tissue resulting in a poor signal-to-noise ratio.

Solutions to High Background Staining

S.No.	Possible Cause	Solution
1	The blocking serum is incorrect	Make sure to block according to the provided protocol.
2	Blocking is insufficient (Do not over-block the tissue because antigenic sites may be masked)	Increase blocking incubation period. Change blocking reagent: For sections: 10% normal serum (1 hr) For cell cultures: 1-5% BSA (0.5 hr)
3	The primary antibody concentration is too high	Titrate the antibody to determine the optimal concentration. Incubate at 4°C.
4	Non-specific binding by secondary antibody	Run a negative control without primary antibody. If you see staining with your secondary only, do one of the following: Change your secondary antibody. Use secondary antibody that has been pre-adsorbed against the immunoglobulin of the species from which your samples were obtained. Block your sample with serum from the same species as the host in which the secondary antibody was raised.
5	Endogenous peroxide or phosphatase is active	Quench the endogenous peroxidase or phosphatase activity by enzyme inhibitors: Peroxidase: H2O2 and methanol (v/v: 0.3%:99.7%). We recommend Boster's 3% H2O2 solution. Phosphatase: 2 mM Levamisole

To reduce background staining further, see our in-depth troubleshooting guide.

#3 Overstaining

Overstaining occurs when an increase in signal reduces contrast, making it difficult to analyze tissue structures accurately. Below are some tips to avoid overstaining.

Troubleshooting Overstaining

S.No.	Possible Cause	Solution
1	Primary antibody too concentrated	Dilute primary antibody solution. Perform a titration to determine optimal antibody dilution.
2	Excessive primary antibody binding	Reduce the length of the incubation step. Incubate in a cold room at 4°C.
3	Detection substrate incubation time too long	Allow less time for signal development after adding the detection substrate.
4	Insufficient washing	Increase the number and time of washes.

#4 Nonspecific Staining

Nonspecific staining occurs when antibodies bind to unintended targets, making it difficult to interpret IHC results. It is often due to improper sample preparation or antibody quality issues.

S.No.	Possible Cause	Solution
1	Inadequate deparaffinization of the tissue section	Increase deparaffinization time. Use fresh dimethyl benzene.
2	Inadequate quenching of endogenous peroxidases or biotins	Use H2O2 to quench endogenous peroxidase activity. Block endogenous biotin with excess free avidin.
3	Insufficient blocking	Increase blocking time.
4	Section dried out	Avoid allowing your tissue section to dry out.
5	Insufficient washing	Increase the washing time and number of washes.
6	Contaminated antibody	Affinity purify your antibodies. Use Boster high-quality antibodies.
7	Excessive primary antibody concentration	Reduce antibody concentration.

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Specimen Preparation: The Foundation of Successful IHC

Proper specimen preparation is critical to maintaining tissue morphology and preserving target epitopes, setting the foundation for any successful IHC protocol. Depending on whether analysis will be done by fluorescence or light microscopy, the preparation methods can vary.

Tissue Collection and Fixation

Tissue must be collected and preserved efficiently to prevent degradation and preserve cell and tissue structure. Two main methods are used:

• Frozen Tissue: Collected in nonaqueous medium, then sectioned in a cryostat at very cold temperatures.
• FFPE (Formalin-Fixed, Paraffin-Embedded): Allows for indefinite storage and room temperature sectioning.

Cryopreservation

After dissection, fresh tissue should be rapidly frozen in chilled isopentane to prevent ice crystal formation. Once frozen, it is stored at -80°C until sectioning. Larger tissues may need to be cut into smaller blocks to ensure even freezing.

Antigen Retrieval

For FFPE tissues, epitopes may become inaccessible due to fixation. Antigens can be unmasked through:

• HIER (Heat-Induced Epitope Retrieval)
• PIER (Protease-Induced Epitope Retrieval)

Both methods aid in restoring antigen accessibility for antibody binding.

Selecting Antibodies for IHC

Choosing the right antibodies is a critical step in ensuring effective IHC results. The following factors should be carefully considered:

• Specificity: It’s essential to select a primary antibody that is highly specific to the target protein. To confirm specificity, it’s best to use tissue samples where the target protein is either knocked out or absent. Other methods, such as observing a single band in Western blotting, can also help verify antibody specificity
• Application Suitability: Antibodies validated for other immunodetection methods, like Western blotting, may not work for IHC, as IHC maintains the native protein conformation. It’s important to confirm that the antibody is validated for IHC to avoid poor staining results.
• Clonality: Monoclonal antibodies recognize a single epitope, offering high specificity, making them ideal for studies requiring precise detection. However, polyclonal antibodies, which bind multiple epitopes, are better suited for detecting proteins present in low abundance, as they offer greater sensitivity but may have more variability.
• Host Source: The host species in which the primary antibody is generated must differ from the tissue being studied to prevent cross-reactivity. When selecting the secondary antibody, it must match the host species of the primary antibody for accurate detection.
• Host Source: The host species in which the primary antibody is generated must differ from the tissue being studied to prevent cross-reactivity. When selecting the secondary antibody, it must match the host species of the primary antibody for accurate detection.
• Blocking and Background Reduction: Non-specific binding can occur if the blocking step is insufficient. To reduce background staining, it’s important to use a blocking serum that matches the host species of the secondary antibody.

Careful consideration of these factors ensures that antibodies perform optimally, resulting in clear, specific staining and accurate data interpretation.

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