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- Table of Contents
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.
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.
Download troubleshooting handbooks for IHC, Western blot and ELISA for FREE.
Troubleshooting guidesDid you know that Boster can accelerate your research projects by assisting you in antibody validation?
Weak 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 |
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1 | Slides lose signal over time during storage |
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2 | The antibody used is not suitable for IHC procedures which detect proteins in its native conformation |
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3 | Fixation procedures (using formalin and paraformaldehyde fixatives) have masked the epitope that the antibody recognizes |
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4 | The primary and/or secondary antibody has lost its activity due to improper storage, dilution, or excessive freezing and thawing |
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5 | Insufficient deparaffinization |
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6 | The protein is located in the nucleus and the antibody cannot penetrate the nucleus |
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7 | The PBS buffer has contaminated with bacteria that damage the phosphate groups on the protein of interest |
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8 | The primary antibody and the secondary antibody are not compatible |
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9 | The protein is not present in the tissue of interest or has not sufficiently expressed |
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10 | Insufficient antibody to detect protein of interest |
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11 | Tissues dried out |
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12 | Enzyme/substrate reaction impeded |
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13 | Buffer incompatible with enzyme |
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14 | Inadequate antigen retrieval |
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High background staining of rat brain tissue using anti-VCP antibody
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 |
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1 | The blocking serum is incorrect |
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2 | Blocking is insufficient (Do not over-block the tissue because antigenic sites may be masked) |
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3 | The primary antibody concentration is too high |
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4 | Non-specific binding by secondary antibody |
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5 | Endogenous peroxide or phosphatase is active |
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6 | Too much amplification (Refer to solution #9 from no/weak staining) |
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7 | Too much substrate was applied (enzymatic detection) |
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8 | Tissue section is not thin enough for reagent penetration |
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9 | Incubation temperature is too high |
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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) |
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11 | Secondary antibody binds endogenous IgG |
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12 | Fixation reagents are still present (due to insufficient tissue washing) |
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13 | Reaction between chromogens and PBS buffer in tissue or cell samples |
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14 | Membrane damage by permeabilization |
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15 | Insufficient deparaffinization |
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16 | High levels of endogenous biotin in biotin-based detection systems for samples (e.g. liver and kidney tissues) |
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17 | Use of polyclonal primary antibody |
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18 | Antibody cross-reactivity |
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19 | Insufficient biotin or lectin blocking |
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Overstaining of mouse liver tissue stained with anti-SC10A1 antibody.
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 |
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1 | Primary antibody too concentrated |
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2 | Excessive primary antibody binding |
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3 | Detection substrate incubation time too long |
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4 | Insufficient washing |
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Nonspecific staining of human tonsil tissue stained with anti-CD3 Epsilon antibody
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 |
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1 | Inadequate deparaffinization of the tissue section |
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2 | Inadequate quenching of endogenous peroxidases or biotins |
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3 | Insufficient blocking |
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4 | Section dried out |
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5 | Insufficient washing |
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6 | Contaminated antibody |
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7 | Excessive primary antibody concentration |
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Unlock new dimensions of biological insight with multiplex IHC! By examining multiple biomarkers simultaneously, our cutting-edge platform allows you to unravel intricate cellular interactions and gain valuable insights for your research.
The principle behind Immunohistochemistry (IHC) entails detection of antigen or happens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Learn more about this in this guide.
Learn the concept of IHC PrincipleBosterBio has a detailed stepwise IHC protocol with a clearly illustrated IHC workflow with recommended reagents. Learn how to effectively implement a successful immunostaining for tissue sections and cell climbing slices.
Learn our IHC ProtocolIHC optimization is a critical step in any test. This guide gives you insight on antigen retrieval, fixation and embedding. Learn how to optimize your immunohistochemistry test to get valuable results.
Check our IHC Optimization tipsLearn the best IHC and ICC sample preparation techniques. Get a detailed procedure of preparing different types of preserved tissues which is key to getting high quality staining during Immunohistochemistry (IHC)..
Check out our IHC Sample Preparation