Fluorescent labels were conjugated initially to antibodies against targets of interest in the 1940s. Since then, several breakthrough advancements have allowed the development of immunohistochemistry.
The advancements include enzyme digestion and heat-driven methods to epitope retrieval, antibody purification and labeling, enhancements in imaging technologies and signal amplification.
Tyramide signal amplification and its benefits
Specifically, both tyramide signal amplification (TSA) and enhancements in imaging have allowed the advancement of immunohistochemistry over conventional or one-color methods to fluorescent multiplex immunohistochemistry (mIHC).
The advancement of TSA allows signal amplification and imaging of low abundance targets by improving the antigen-associated fluorescence signal.
In combination with the availability of a huge number of fluorophore filters and reporters, this allows a more precise visualization of a huge number of targets of interest inside a single tissue sample. At the same time, enhancements in software and imaging platforms have allowed high quality and data-dense images to be captured.
Regardless of these developments, more extensive use of fluorescent mIHC with TSA is restricted by the time needed to carry out the staining, specifically when several targets of interest are investigated. Process automation might reduce the hands-on time needed to carry out the method and increase throughput.
But such instrumentation is usually cost-intensive and does not avoid the need for considerable time investment up-front in the form of validation and optimization.
Likewise, multispectral imaging of entire slides at high resolution also takes a lot of time. The software and equipment needed to capture high resolution images are also costly, which further restricts the number of users.
Although a majority of the previous utility of IHC was limited to the basic science laboratory, the advancements mentioned in this article have made fluorescent mIHC using TSA into a robust tool for translational research.
Going forward, more technical advancements and more extensive availability and affordability of imaging technology will probably enable mIHC transition to a combined, workhorse clinical tool.
Clinical use will necessitate highly reproducible, measurable outcomes, as well as adherence to regulatory needs. Clinical mIHC applications will probably have to be developed with guidance from organizations like the American Society for Clinical Pathology.
But these applications exhibit huge potential for achieving considerable progress in clinical patient care, such as characterization of the complex tumor microenvironment and a further selection of targeted therapies.
Figure 2. Detection of human CD8 (green) TIGIT (red) and PD-L1 (cyan) in breast carcinoma by IHC-IF. Antibodies: Rabbit anti-CD8 recombinant monoclonal [BLR044F] (A700-044), rabbit anti-TIGIT recombinant monoclonal [BLR047F] (A700-047) and rabbit anti-PD-L1 recombinant monoclonal [BLR020E] (A700-020). Secondary: HRP-conjugated goat anti-rabbit IgG (A120-501P). Substrate: Opal™ 520, Opal™ 690, and Opal™ 480. Counterstain: DAPI (blue). Image Credit: Bethyl Laboratories Inc.
Figure 3. Detection of human CD3 (green), CD20 (magenta), CD68 (yellow), and cytokeratin (red) in FFPE tonsil by IHC-IF. Antibodies: Rabbit anti-CD3E recombinant monoclonal [BL-298-5D12] (A700-016), mouse anti-CD20 monoclonal [L26] (A500-017A), mouse anti-CD68 monoclonal [KP-1] (A500-018A), and mouse anti-Cytokeratin [AE1/AE3] (A500-019A). Secondary: HRP-conjugated goat anti-rabbit IgG (A120-501P) and HRP-conjugated goat anti-mouse IgG (A90-116P). Substrate: Opal™. Counterstain: DAPI (blue). Image Credit: Bethyl Laboratories Inc.
Figure 4. Detection of human CD3 (aqua), CD8 (green), cytokeratin (red), and PD-L1 (magenta) in FFPE lung carcinoma by IHC-IF. Rabbit anti-CD3E recombinant monoclonal [BL-298-5D12] (A700-016), rabbit anti-CD8 alpha [BLR044F] (A700-044), mouse anti-Cytokeratin [AE1/AE3] (A500-019A), and rabbit anti-PD-L1 [BLR020E] (A700-020). Secondary: HRP-conjugated goat anti-rabbit IgG (A120-501P) and HRP-conjugated goat anti-mouse IgG (A90-116P). Substrate: Opal™. Counterstain: DAPI (blue). Image Credit: Bethyl Laboratories Inc.
About Bethyl Laboratories, Inc.
Bethyl Laboratories, Inc. has been dedicated to supporting scientific discovery through its qualified antibody products and custom polyclonal antibody services since its founding in 1972.
Currently, Bethyl’s portfolio consists of over 9,500 catalog products; offering over 8,200 primary antibodies targeting over 3,300 proteins and 1,300 secondary antibodies raised against immunoglobulins from over 25 species.
With over 45 years of experience, Bethyl is also a leading provider of custom polyclonal antibody production services. Bethyl offers complete packages from initial peptide synthesis to affinity purification of custom polyclonal antibodies from an antigen-specific immunosorbent.
Most recently, our capabilities have expanded and we now offer contract recombinant rabbit monoclonal antibody partnerships.
Every antibody that Bethyl sells has been manufactured to exacting standards at its sole location in Montgomery, Texas, and has been validated in-house by Bethyl’s team of scientists. From the veterinary facilities to the development, production and validation labs, the entire Bethyl team focuses on delivering quality products.
What does it take for an antibody to pass Bethyl’s validation and quality control? In other words, what makes a really good antibody? First and foremost, an antibody must be shown to recognize the intended protein target. An antibody must also show high sensitivity with minimal cross-reactivity.
To achieve these goals, Bethyl has devised a unique process for validating the specificity of its antibody products, which is critical to reproducibility of antibody-based experiments in life science.
While many companies settle on using data from a single antibody, Bethyl tests with paired antibodies raised against distinct protein epitopes.
Only after an antibody has been validated for specificity in immunoprecipitation and/or Western blot is it then tested in additional applications such as immunohistochemistry, immunocytochemistry, ChIP and proximity ligation assay.
Because of its rigorous validation process and high standards, Bethyl does not sell every antibody it makes. Bethyl serves to advance science by concentrating its resources on developing qualified antibodies, including many to emerging and underserved protein targets.
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