Headphones on, I am listening to my favorite music and staring at the lab bench in front of me feeling excited to start the immunohistochemistry experiment of the day. Pipette, timer, wells, vials, brush, buffers. Protocol taped on the shelf. Everything is ready. I feel like a DJ at the console, and I find myself thinking: what makes a good DJ? A DJ does not just play one song after the other in a random sequence. He has everything under control, he plans and underpins, every minute of a performance to varying degrees. So, how do I get started when planning a new set for a forthcoming immunohistochemistry experiment? Read on to find out how to control your results and prepare for a successful ’gig’ in the lab.
Control is key!
Whether you attend a live rock concert, or you join a friend at a live DJ’s performance at your favorite club, you know exactly what to expect. Hours of enjoyable music harmoniously blended together to shape a final mix. Fine adjustments and levels balancing to make seamless track transitions. However, what you see is the DJ scratching and dancing on the stage, playing with the mixer and the turntable set. It might seem that he is letting them do all the work, that he is improvising, just randomly making things up as he goes along. But this has nothing to do with luck.
Control your IHC like a DJ
If you want to perform like a DJ, then you need to have everything under control. All variables must be carefully set and monitored throughout your experiment. In immunohistochemistry a controlled experiment is one in which everything is held constant except for one variable. Seems impossible? It only requires a few simple steps and a little bit of effort.
Controls for IHC
In science, experimental controls eliminate alternative explanations of experimental results especially experimental errors and experimenter bias. In immunohistochemistry, controls are necessary for the validation of staining results. Without them, the interpretation of any staining would be unreliable, and the results would be less valuable. More specifically, controls help determine if the staining protocols were followed correctly, whether day-to-day or worker-to-worker variations have occurred and indicate if the reagents performed flawlessly throughout the experiments.
But how do you select the proper controls? The task to select and apply the correct controls is a daunting task. Selection of appropriate controls is not purely a technical issue. It requires in-depth knowledge of how the test will be used.
Internal and external controls
Most often, controls are specific to the type of experiment being performed (these are referred to as internal controls); other types of control have the job of ensuring that the equipment is working properly (referred to as external controls). Both internal and external controls are essential in monitoring the reproducibility of the IHC method that you are performing.
- Internal controls: provide an indication if your test was successful or not. It reflects success or failure of the preanalytical and analytical stages and components, such as tissue processing, fixation, antibody specificity and dilution, buffers and reagents.
- External controls: control the analytical and post-analytical phase of your IHC protocol, such as development, visualization, and quantification of the staining.
Positive and negative controls
- Positive controls: tissues or cells containing the target antigen in its known location such as in a specific cell type or in a specific intracellular compartment. The positive control confirms that your target antigen is expressed by the relevant cells and tissues. With a well characterised positive control, a known response should be expected. However, in IHC the interpretation of positive controls can be problematic, particularly in the case of antibodies that are directed against targets that are expressed ubiquitously or at very low levels.
Example of positive controls are normal tissues or cancer tissues that are known to express the protein of interest. They are typically used to show that the IHC staining is successful and capable to detect the target of interest.
In case where there are two possible outcomes, e.g. positive or negative staining, a positive result in the positive control strongly indicates that the immunostaining has been successful. A negative result in the positive control indicates failure of the IHC procedure.
- Negative controls: consist of tissues or cells where the target protein is known to be absent. The negative control group is one in which no response is expected. Negative controls assist in the identification of positive errors.
Example of negative controls are buffers and reagents lacking antibodies applied in the staining reaction typically used to validate the specificity of the primary and secondary antibodies, in order to ascertain that the antibody-antigen reaction is exclusively due to the expression of the target of interest.
In case where there are two possible outcomes, e.g. positive or negative staining, a positive result in the negative control indicates failure of the IHC procedure. A negative result in the negative control strongly suggests that the IHC procedure has been successful.
Cell lines and normal- and cancer tissues, transfected cells, Wester blots, studies on transgenic animal models or cell lines over-expressing or being knocked-down or knocked-out for a certain protein of interest, can be used to run positive and negative controls.
Take a look at the example illustrated in the picture below. As predicted, human small intestine shows high expression of ACE protein (positive control), while the staining of human liver shows low expression of ACE protein as expected (negative control).
Figure 1. Immunohistochemistry analysis in human small intestine and liver tissues using Anti-ACE antibody (HPA029298). Corresponding ACE RNA-seq data are presented for the same tissues.
All controls required in immunocytochemistry (internal and external, positive and negative controls) can be applied at three different levels:
- Primary antibody-related controls that show the specificity of the primary antibody binding to the antigen.
- Secondary antibody-related controls that show the label is specific to the primary antibody.
- Tissue-related controls that show the labeling is the result of the label added and not the result of tissue morphology.
Let them know
When preparing a manuscript, it is critical that the controls used are listed in the methods section to demonstrate to the reviewers and readers that the authors were aware of the controls and that they performed them. Not only must these controls convince the scientist doing the experiments, but they also must convince the reader that the results are correct.
The prerequisites for controls in immunohistochemistry are now well known, not only for publication and data reproducibility. By the time of publication, the reviewer and readers should be convinced that proper IHC controls and data analyses have been done.
The editors and reviewers must make an effort to ensure that the guidelines to authors are strictly followed. The compilation of a checklist on how to report the results of an IHC experiments will greatly benefit the spotting of omissions.
Consider the simple points outlined in the figure below. You can use it as an easy checklist to validate the IHC results reported in your publication.
Why are controls important?
Simply stated, immunohistochemical results that lack appropriate controls cannot be properly interpreted.
In the last couple of decades there has been an exponential increase in the number of publications on immunohistochemistry and immunocytochemical techniques. Hence, standardization of controls for every step is crucial for the achievement of reproducible and reliable results.
Immunohistochemistry has to an increased degree become an indispensable technique within anatomic pathology laboratories. Imagine the responsibility of selecting and monitoring immunohistochemistry controls for a pathologist who will be relying upon these results in his diagnostic work.
Fast rewind - how do I choose the right controls?
In a perfect world with unlimited time and resources, every control should be performed, and every condition matched perfectly. But this is not a realistic scenario especially when working with human tissues.
I recommend that you run at least the following controls at the start of a new protocol:
- Test the antibodies specificity in your application.
- Perform each staining with primary antibody by itself.
- Incubate tissue or cells with secondary antibody only.
- Examine each primary antibody with the secondary from another species.
- Use negative controls: cells or tissue that lack the protein of interest.
Unleash your DJ-ing powers: it is show time!
Do you have your control under control and your compilation of slices to stain? Everything is ready now. Pipette, timer, wells, vials, brush, buffers. Protocol taped on the shelf. Are you ready to start? Great! Put your headphones on. Set the radio on your favorite music station and start pipetting!
If you are looking for human tissue sections to use as positive or negative control in your immunohistochemistry experiments explore the Tissue Atlas section of the Human Protein Atlas. In our previous blog post, you can learn all about how use the Tissue Atlas. Read on to learn more!
Now you have an idea of how to control your IHC controls. The next step is to know which primary antibody controls you need.
Continue to our next blog post "Control Your IHC: Primary Antibody Controls You Should Know" to learn more.
Brooks HL. and Lindsey ML. (2018) Review: guidelines for authors and reviewers on antibody use in physiology studies. Am J Physiol Heart Circ Physiol. 314:H724-H732.
Burry RW. (2011) Controls for immunocytochemistry: an update. J Histochem Cytochem. 59(1):6-12.
Hewitt S.M. et al. (2014) Controls for Immunohistochemistry. J Histochem Cytochem. 62(10): 693–697.
Torlakovic EE, et al. (2014) Standardization of negative controls in diagnostic immunohistochemistry: recomandations from the international Ad Hoc expert panel. Appl Immunohistochem Mol Morphol. 22(4): 241-252
Torlakovic EE, et al. (2015) Getting controls under control:the time is now for immunohistochemistry. J Clin Pathol. 68:879-882
Image credit, top image: Photo by Gavin Whitner (MusicOomph.com).