Immunohistochemistry (IHC) Troubleshooting Guide

We provide tables about common issues with IHC staining: weak staining, high background, overstaining, nonspecific staining. For more in-depth troubleshooting tips, please download our ebooks below.

How to Troubleshoot IHC

The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the IHC assays.

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

If you do not see the issues you are having featured in this page, please contact us at [email protected] and we will help you resolve your specific trouble.

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No Or Weak Staining

Weak staining of CD3 epsilon in human tonsil tissue

Weak staining of CD3 epsilon in human tonsil tissue

Improved staining of CD3 epsilon in human tonsil tissue

Improved staining of CD3 epsilon in human tonsil tissue

Good results in IHC experiments depend on strong, specific staining of the target antigens. A good result can only be achieved when a sufficient quantity of primary antibody penetrates the sample and binds its target with high specificity, and enough secondary antibody with active enzymatic or fluorescent conjugate binds the primary antibody. When an IHC experiment results in faint or weak signal, it often has to be repeated, costing valuable time, money, and resources. Use the troubleshooting guide below to identify and resolve the source of your weak signal.

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.

High Background

High background staining of rat brain tissue using anti-VCP antibody

High background staining of rat brain tissue using anti-VCP antibody

Improved staining of VCP in rat brain tissue

Improved staining of VCP in rat brain tissue

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, steps must be taken to reduce it. Background staining can be caused by inappropriate antibody binding or by mistakes during the preparation of the tissue slide. Use the guide below to resolve your 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:
    1. For sections: 10% normal serum (1 hr)
    2. 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
6 Too much amplification (Refer to solution #9 from no/weak staining)
  • Reduce amplification incubation time.
  • Dilute the secondary antibody.
7 Too much substrate was applied (enzymatic detection)
  • Further dilute substrate to reduce substrate concentration.
  • Reduce substrate incubation time.
  • Choose substrate of higher S/N ratio (e.g. metal-enhanced DAB).
8 Tissue section is not thin enough for reagent penetration
  • Prepare thinner sections.
9 Incubation temperature is too high
  • Incubate samples at 4°C.
10 Primary antibody was raised in the same species as source of tissue (therefore, secondary antibody recognizes and binds to everywhere on the entire tissue because it was raised against that species)
  • Use primary antibody raised against a species which is different from the source of tissue.
  • Use biotinylated primary antibody and conjugated streptavidin for the detection system.
11 Secondary antibody binds endogenous IgG
  • Include control slide stained without the primary antibody to confirm whether the secondary antibody is the source of the background.
12 Fixation reagents are still present (due to insufficient tissue washing)
  • Wash the tissues extensively with PBS buffer.
13 Reaction between chromogens and PBS buffer in tissue or cell samples
  • Before incubating with the substrate, use Tris buffer to wash the samples.
14 Membrane damage by permeabilization
  • Use a less stringent detergent such as Tween 20 (instead of Triton X).
  • Remove permeabilizing agent from your buffers.
15 Insufficient deparaffinization
  • Increase the deparaffinization time.
  • Use fresh dimethylbenzene.
16 High levels of endogenous biotin in biotin-based detection systems for samples (e.g. liver and kidney tissues)
  • Perform biotin block after normal blocking procedure (before primary antibody incubation).
  • Use polymer-based detection.
17 Use of polyclonal primary antibody
  • Use monoclonal primary antibody to reduce cross-reactivity.
18 Antibody cross-reactivity
  • Use Boster primary antibodies guaranteed specific to only their listed targets.
19 Insufficient biotin or lectin blocking
  • Block endogenous biotin with streptavidin solution.
  • Block endogenous lectins with alpha-methyl mannoside buffer.

Overstaining

Overstaining of mouse liver tissue stained with anti-SC10A1 antibody

Overstaining of mouse liver tissue stained with anti-SC10A1 antibody.

Improved staining of VSC10A1 in mouse liver tissue

Improved staining of VSC10A1 in mouse liver tissue

Overstaining occurs due to excessive development of signal in the sample. This causes the sample to become saturated, reducing contrast. Overstained samples can appear blurry, diffuse, or monochromatic. Overstaining can prevent accurate visualization of tissue structures and inhibit useful detection of protein localization. Below are some tips to reduce overstaining when performing IHC.

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.

Nonspecific Staining

Nonspecific staining of human tonsil tissue stained with anti-CD3 Epsilon antibody

Nonspecific staining of human tonsil tissue stained with anti-CD3 Epsilon antibody

Improved staining of CD3 Epsilon in human tonsil tissue

Improved staining of CD3 Epsilon in human tonsil tissue

Immunohistochemistry (IHC) is one of the many methods that researchers use to visually detect specific antigens in a sample. A variety of issues can arise during the staining step of IHC, such as non-specific staining. Non-specific staining occurs when the primary antibodies bind to proteins other than the target protein, resulting in data unusable for meaningful interpretation. There are two main causes of non-specific staining in IHC - improper sample preparation and antibody problems.

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.