Bio-Rad AbD Serotec Ltd
Product Manager Antibody Reagents
Bio-Rad AbD Serotec Ltd
Development Scientist
Immunohistochemistry (IHC) is routinely used in diagnostic studies to determine morphological abnormalities and the presence of biomarkers indicative of certain diseases such as cancer. An IHC experiment utilizes a primary antibody that binds specifically to a protein of interest present in a tissue sample. The antibody binding event is visualized by a detection system, which provides information about the presence and location of the protein in the tissue.
Since the first IHC experiments in the 1940s, much progress has been made in terms of developing more sensitive detection systems. Early on, secondary antibodies conjugated to enzymatic labels were exclusively used for visualization. To further improve on the methodology, IHC kits utilizing polymer/dextran or other amplification reagents and fluorescent labels have been developed. The large number of fluorescent labels has improved the options for researchers to detect several antigens simultaneously on the same tissue specimen, a technique known as multiplexing. Multiplex IHC provides a greater level of detection and understanding of protein localization, interactions and cellular regulation.
Selection of the appropriate antibody reagents for multiplex-IHC experiments can be challenging as there are many variables that need to be considered to gain maximal detection and to avoid staining artifacts. These may include the staining method (sequential vs. simultaneous), undesired species cross-reactivity of primary and secondary antibodies, and antibody format/type availability. Each of these parameters is important to the success of the experiment and should be researched and/or optimized ahead of time. This article will focus on several hands-on tips for selecting enzymatic or fluorescent-label combinations for IHC-multiplexing experiments.
IHC multiplexing with multiple enzymatic labels
Multiplex-IHC experiments enable researchers to study the co-localization of two or more proteins. Compared with single-IHC experiments, there are the potential advantages of gathering more information from the same sample, faster time to results and potential cost savings when performing multiplex IHC experiments.
When performing a double staining for two proteins that localize in the same sub-cellular compartment, be aware that after the first chromogenic precipitate (the result of the reaction of the enzymatic label with its chromogenic substrate) has stained/saturated the compartment it becomes very difficult for the second one to stain the same compartment adequately. Designing experiments with multiple enzymatic labels also has its challenges because of the limitations of detection-color combinations. As a general rule, all experiments should include suitable counterstains and the final precipitate colors should be fairly easy to differentiate spectrally [1].
Here are some suggested double-staining combinations that have been demonstrated to work effectively on a variety of tissue samples [2,3]:
- Turquoise-red: Beta-galactosidase/X-Gal (turquoise), alkaline phosphatase (AP)/Fast Red (red); use methyl green as the counterstain.
- Blue-red: AP/Fast Blue BB/Naphthol-AS-MX-phosphate (blue), horseradish peroxidase (HRP)/3-amino-9-ethylcarbazole (AEC) (red); a potential counterstain is methyl green. It has been reported that methyl green can bind to the red HRP/AEC reaction product, which can result in a color shift from red to brown [3].
- Red-brown: AP/Liquid Permanent Red (LPR) (red), HRP/3,3'-Diaminobenzidine (DAB) (brown); use blue nuclear hematoxylin as the counterstain.
When looking at co-localization, one has to be able to differentiate each of the single stains from the merged results. Therefore, designing triple-staining experiments is highly challenging as each color needs to be unambiguously identifiable. Potential enzyme and chromogen triple-staining combinations are shown below [4]:
- Turquoise-blue-red: Beta-galactosidase/X-Gal (turquoise), AP/Fast Blue (blue), HRP/AEC (red).
- Brown-turquoise-red: HRP/DAB (brown), Beta-galactosidase/X-Gal (turquoise), AP/Fast Red (red).
- Brown-red-green: HRP/DAB (brown), AP/New Fucsin (red), HRP/3,3′,5,5′-Tetramethylbenzidine (TMB) (green).
However, with the availability of powerful multi-spectral imaging systems, such as the Nuance VIS-FL Multispectral Imaging System from Cambridge Research Instrumentation, which enable the separation of visually identical-appearing precipitates, the selection criteria for enzyme-chromogen combinations have changed [5]. These days, chromogens are no longer purely selected for their color but also for their staining efficiency, intensity, (resulting in crisp, well-defined staining patterns) as well as being able to be mounted organically [2,3].
IHC multiplexing with multiple fluorescent labels
When designing multi-color imaging experiments it is crucial to first model the excitation and emission spectra of all fluorophores intended to be used [6]. This can be performed quickly, using various free fluorescence spectral analyzer software programs available on the websites of antibody and microscope manufacturers. The focus of such mock-ups is to ensure that there is minimal emission-spectra overlap between the selected fluorophores. Such overlaps could lead to a phenomenon known as “bleed-through”, which results in the detection of a fluorophore's emission in another fluorophore's filter set. To reduce this risk, use fluorophores with narrow emission spectra and carefully select the microscope filters to closely match the excitation and emission spectra. It is important to check for actual bleed-through by including single-labeled control samples in the experimental design and observing these in the filter sets of the other fluorophores.
Another tip when staining with multiple fluorophores is to visualize the least abundant protein with the fluorophore with the highest quantum yield and to visualize the most abundant protein with the dimmest fluorophore. If two or more similarly abundant proteins are desired to be detected, it is possible to reduce the concentration of the antibody conjugated to the fluorophore with the shortest wavelength to avoid bleed-through into longer wavelength fluorophores. Another option is to balance the exposure/gain/neutral density filter settings to reduce the intensity of super-bright fluorophores.
As described for enzymatic labels, the availability of spectral unmixing systems has also eased the design of experiments with multiple fluorophores by allowing the separation of fluorophores with extensive spectral overlap.
Multiplex solutions
Multiplex IHC is a powerful and informative technique to study co-localization of multiple proteins. Improvements to the reagents and detection methods have enabled researchers to better characterize their protein targets. As with every experiment, appropriate controls should always be included. For multiplex experiments we highly recommend autofluorescence controls, single staining controls and secondary antibody-only controls. Following these suggested tips and considerations when designing and performing your multiplex-IHC experiments will lead to exciting and novel discoveries.
References
[1] AbD Serotec, IHC Tip 4: Which counterstains are most compatible with my antibody staining? 2015.
[2] Van der Loos, C.M., "User Protocol: Practical Guide to Multiple Staining," 2008.
[3] Van der Loos, C. M. Chromogens in multiple immunohistochemical staining used for visual assessment and spectral imaging: the colorful future. The Journal of Histotechnology, Vol. 33, No. 1, 2010.
[4] Christensen, N, K, Winther, L, Multi-staining immunohistochemistry, Chap 15 IHC guidebook, 5th edition, 2008.
[5] Van der Loos, C. M., "Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging". J Histochem Cytochem, 56(4): 313–328, April 2008. [PubMed ID: 18158282]
[6] AbD Serotec, 10 Tips for Selecting and Using Fluorophores in IF Experiments. 2015.
Image: Rabbit anti-MCM2 (AHP1522 + STAR36D549) + Mouse anti-CD21 (MCA1808 + STAR74D488) on paraffin human tonsil, AbD Serotec.
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