How many decisions do you think you make every day? Would you be surprised if I tell you that an adult person makes about 25,000 more or less conscious decisions every day? It’s a fact. Consciously or unconsciously, we spend every day of our lives making decisions: where to go, what to wear, what to eat, how to spend our time, etc. But how much time do you spend making a decision for the right control for your primary antibody? And, most importantly, did you make the right choice? How can you tell a good decision from a bad one?
Sometimes making decisions is simple. In many cases, the consequences are seldom that important. But some decisions carry more weight than others. No matter how easy or tough a decision is, the strategy you adopt, as well as the resources of time, talent and energy you devote to take that decision, will likely affect the outcome.
Controls constitute an ubiquitous part of a well-designed scientific experiment. The selection of proper controls for immunohistochemistry (IHC) is essential to get reliable and reproducible results as a basis for good scientific practice. Unfortunately, there are no controls to control everything.
Control Your IHC At Three Different Levels
In our previous post we discussed internal and external, positive and negative controls for IHC.
We mentioned that IHC controls can be applied at 3 different levels:
- Primary antibody controls: these controls show the specificity of the primary antibody towards the antigen of interest and help evaluate the stringency of the IHC protocol.
- Secondary antibody controls: these controls show that the label is indeed specifically associated with the primary antibody.
- Tissue/cell-related controls: confirm the immunostaining is the result of the label added and not due to endogenous staining artefacts arising from the sample itself.
All three types of control provide support that the readout of the IHC staining is reliable. In other words, they indicate that what you see in the microscope is the true presence of the antigen.
In this post, we focus our attention on the primary antibody-related controls.
Importance Of Primary Antibody Controls
As mentioned above, the intent of primary antibody controls is to verify the specificity of the primary antibody binding to the antigen of interest. In addition, controls for the primary antibody are applied to confirm the correctness of the preanalytical IHC phase related to sample preparation, such as fixation, post fixation, storage and processing. Primary antibody controls must be included for each new antibody, protocol and application that you test.
Primary Antibody Controls You Should Know
A wide range of methods are available to control the primary antibody binding to the target antigen and the accuracy of the IHC protocol. The lack of a single widely applied method for this purpose confirms the challenge of demonstrating this point.
If you are familiar with the concept of antibody validation you will recognize some of these methods (e.g. western blot, siRNA and double labelling). If you want to know more about antibody validation read our previous blog post here.
Read on for a summary of the methods used for primary antibody control applicable to IHC regardless of the labeling method.
1. Genetic Methods Used For Primary Antibody Control
- Recombinant Expression: A method used for a primary antibody control is the use of a transfected cell line recombinantly expressing the protein recognized by the primary antibody. The untransfected cells serve as a negative control since they do not endogenously express the protein of interest. Confounding factors or limitations: low transfection yield and misexpression due to poor cell host selection.
- In-situ hybridization: Its utility rests on the concept that a given mRNA expression co-occurs with the corresponding translation product (protein) at the cellular level. The recent and revolutionary development of branched DNA technologies, like RNAScope, has now enabled the application of in-situ hybridization on FFPE-tissues to provide for mRNA expression equivalent to immunohistochemistry protocols. Confounding factors or limitations: the expression of the protein can be distal from the site of mRNA-production within the cell (e.g. in neurons). Some transcripts may also have a very short turnover time within the cell prior to translation. Furthermore, some proteins may be less stable than their transcripts.
- CRISPR (knockout) / siRNA (knockdown): these represent approaches for guided removal or reduction of a protein within a cell and non-human in vivo models allowing the tissue to be fixed and prepared using the same procedure as the experimental animals. For CRISPR-based techniques, the gene expression of a protein is completely and permanently erased. With siRNA the reduction of gene expression is temporary. If you work with an antibody with high specificity for the gene products knocked out, no immunostaining should be detected. Confounding factors or limitations: these methods are only applicable for cells and animal models, not for human tissues.The success of these methods is also dependent on the genetic constructs and the transfection efficiency. Another confounding factor relates to the incompatibility of a knockout with cell viability.
2. Immunogenic Methods Used For Primary Antibody Control
Western blot can be used to show that the antibody labels a single band representing protein with the correct known molecular weight. These immunoblots are technically straightforward and thus the most common specificity control applied for commercial antibodies. This method as well has its flaws. Confounding factors or limitations: the protein is unfixed but denatured in SDS, losing its secondary and tertiary structure. If the immunogen is a full-length protein, nonspecific binding may occur within the tissue, causing erroneous interpretation of the IHC staining. The differences in sample handling between western blot and IHC could affect the epitope in different ways.
3. Double Labelling used for Primary Antibody Control
Another method for primary antibody control is through colocalization with an additional antibody or other affinity reagent showing binding to the same target protein albeit at a different binding site. Confounding factors or limitations: difficulties in the subcellular resolution of the immunostaining due to steric hindrance. Lack of available second primary antibody which may necessitate an orthogonal method like RNA-ISH or use of a ligand-binding assay.
4. Adsorption Control
Enzyme-linked immunosorbent assay (ELISA) is often employed as a method for antibody validation. Prior to labeling, the primary antibody is mixed and absorbed with the purified antigen in a tube. The antibody binds with the antigen, gets absorbed and is hence no longer available to bind to antigens within the section. As a result, no staining should be visible. Confounding factors or limitations: the absorption control does not exclude the binding of the primary antibody to proteins other than the one used for incubation. It only shows the specificity of the antibody for the antigen used for the absorption.
5. Isotype Control
An isotype control is an antibody of the same isotype (e.g. IgG1, IgG2 etc.), clonality, conjugation, and host species as the primary antibody. This allows for the simultaneous application of two or more primary antibodies from the same host in the same sample as long as they each belong to different isotypes. Since the secondary antibody only will bind one isotype, no cross-reactivity should occur. Confounding factors or limitations: limited availability of primary antibodies with different isotypes. These controls are only applicable to verify the specificity of mouse monoclonal primary antibodies (mAbs).
Do Not Get Fooled
As for negative controls of the primary antibody staining, many scientific publications report on immunohistochemical assays performed with precisely the same protocol and the identical tissues simply by omission of the primary antibody. Do not get fooled by this flawed method! The absence of staining when omitting the primary antibody is merely a control for nonspecific binding of the secondary antibody and not the evidence for the specificity of staining with the primary antibody.
An appropriate negative control for primary antibody staining is, rather, the substitution of the primary antibody for serum or isotype-specific immunoglobulins at the same protein concentration as the primary antibody.
How Do I Choose The Right Control For My Primary Antibody?
The selection of appropriate controls is not a purely technical issue. It is also about selecting a relevant/desirable calibration set appropriate for a particular experimental or clinical use. It requires in-depth knowledge of how the tests are used. General recommendations when choosing your primary antibody control:
- Select appropriate positive and negative tissue controls and, if relevant and available, tissues from knockdown/knockout animals.
- If possible run your IHC with two different primary antibodies recognizing two separate epitopes for the same antigen.
- Absorption controls are a good choice; however, they should be used with caution and in combination with other controls.
- If required use several orthogonal methods, like RNA ISH, to show the specificity of the primary antibody.
If you are looking for human tissue sections to use as positive or negative control in your immunohistochemistry experiments explore the Tissue Atlas or read our previous blog post here.
Download our table guide “Primary antibody controls: principles, advantages and limitations”. Make sure to read it before planning your next experiment.
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