How to Detect and Solve IHC Issues: A Guide

Do you find it difficult to get your antibody to work during IHC experiments? For a successful experiment, it is essential to use an antibody that has been officially validated for IHC.

Validating Your Antibody

Here, we present a few important questions to ask when evaluating an antibody:

  • Examine the validation data and images from the antibody supplier. Check if the antibody has been evaluated for IHC on the tissues that are currently being used.
  • Check if the antibody has been tested in multiple tissues, in order to provide important information about how specific the antibody is and how it can perform across many samples.
  • Check if the specificity of the antibody has been further validated by an enhanced validation method. A possible enhanced validation method is to compare the antibody staining with a non-antibody-based method. This can be applied in IHC by comparing the antibody staining with RNA-Seq data for the same tissues. The antibody specificity is confirmed when the antibody signal in IHC correlates with the RNA levels.
  • In the Nature Methods article "A proposal for validation of antibodies" (Uhlén et al 2016), there is more information about methods for enhanced validation.
  • During the process of antibody validation, the application and context are definitive, so it will need to be verified that the antibody works as expected in the specific setup, even if the supplier has validated the antibody.

Knowing the Expression of Your Target Protein and the Importance of Controls

Even though it may be evident, it is still crucial to make sure that the protein in the study is sufficient in amount to be detected in the tissues that are used.

It is also necessary to include appropriate tissue controls, making sure to use both positive and negative tissues.

A positive control is an indicator that the protocol is working. The tissue used as positive control should be noted to express the target. The negative control indicates the specificity of the antibody. The negative control tissue should not express the target. As a matter of choice, knockout or knockdown system can be used. ​

Below is the best advice to help find information about protein expression and suitable control tissues:

  • Look at the product images found on the supplier's product page.
  • Study product references on the supplier's product page.
  • Use the Human Protein Atlas, where the protein and RNA expression levels in a number of human tissue types, tumors and cell lines are presented.
  • Learn about the protein in databases such as Uniprot and GeneCards. PubMed is also recommended. Check what the literature says about where the protein should be expressed and where not.
  • Check with the supplier's support, where help is always provided by the support team.

Tips and Tricks for Optimizing the IHC Experiment

Once the validation of the antibody has been confirmed, it is time to optimize the protocol. Below are the things that have to be considered and some of the most common advice on how to do this more easily:

  • It is always a good start using the protocol recommended by the supplier as closely as possible.  The protocols recommended by Atlas Antibodies can be found here.  

Tissue Treatment

The way tissue samples are treated is correlated to the IHC results that can be achieved. Fixation and dehydration are important steps that, if not performed/optimized correctly, can affect the morphology and the overall staining result.

  • It is important that the fixation starts instantly after dissecting/grossing.
  • Sample thickness, fixative/tissue ratio, time and volume of the fixative are crucial steps to consider when optimizing.
  • The water present in the cells during the dehydration step has to be removed, since this may cause the tissue sample to shrink, making it too dry and crisp.

Antigen Retrieval

Cross-links are formed during the fixation step and it is important to break those, which is made possible by unmasking the epitopes with antigen retrieval. The optimal antigen retrieval for each target, tissue and antibody needs to be optimized.

  • Adjusting different parameters such as heat, pH, buffer, retrieval time and temperature can be helpful in the optimization of the setup. See the IHC protocol for suggestions.
  • Even if optimized, the effectiveness of the antigen retrieval step is variable and some epitopes can remain hidden. Polyclonal antibodies can be an advantage since they recognize several epitopes.  

Reagents

The stability and binding properties of the antibody can also be affected in various ways by buffers and diluents. Therefore, they need to be optimized and adapted for the antibody and setup.

  • The product data sheets provide information about the recommended dilutions for both the primary and secondary antibodies. These dilutions are a starting point, but need to be optimized for  the setup and protocol.
  • TBS buffers are preferable in order to avoid non-specific staining.
  • PBS buffers can reduce the specific binding abilities of the antibody. Some antibodies can bind to phosphates in the PBS buffer instead of the target protein giving a false negative or weak staining.  
  • To reduce non-specific binding, higher concentrations of salts can be added (e.g. sodium chloride) and detergents (e.g. Tween 20) to the wash buffers.
  • To reduce nonspecific staining, it is important to remember the appropriate blocking experiments, like protein blocking, biotin blocking and blocking for endogenous enzymes (alkaline phosphatase and peroxidase).

About Validation at Atlas Antibodies and the Human Protein Atlas

Atlas Antibodies have always worked extensively with antibody validation and with producing highly characterized antibodies that customers can trust. The antibodies are developed by the Human Protein Atlas and are strictly evaluated for specificity and performance. They are also characterized in several applications. They are validated for IHC in all major tissues and organs as well as the 20 most common cancer types.

A second layer of validation, enhanced validation, is being introduced to the antibodies, following the Human Protein Atlas' interpretation of the recommendations from the International Working Group for Antibody Validation (IWGAV) published in Nature Methods.

The Scientific Support of Atlas Antibodies can provide further help and advice about antibody validation.

Atlas Antibodies

At Atlas Antibodies we have a very clear mission: To provide our customers with advanced research reagents targeting all human proteins.

The Human Protein Atlas (HPA) project released the first version of a complete tissue-based map of human protein expression using antibodies in November 2014. In close partnership with the HPA project, we continue to develop advanced antibodies and advanced reagents for Mass Spectrometry (MS)-based quantitative proteomics.

Of the possible 20,000 protein coding genes in the human body we already have over 21,000 antibodies covering 15,000 gene products and an additional 20,000 protein quantification MS-standards representing 13,000 protein targets. Learn more in this short film about us.


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Last updated: Aug 29, 2019 at 10:52 AM

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