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By Rosa Moreno, PhD.
This new introductory guide to methods and techniques in immunohistochemistry is written by Dr. Alexander E. Kalyuzhny, Bio-Techne, for the Springer series: Techniques in Life Science and Biomedicine for the Non-Expert . This guide provides a comprehensive introduction along with conceptual and methodological basics for new users to ensure a clear understanding upon which researchers may build their expertise in immunohistochemistry approaches.
Immunohistochemistry techniques harness the intrinsic specificity of antibody-antigen binding to detect, localize and quantify proteins of interest within tissues. The origins of immunohistochemistry date back to the 1940s and the technique emerged from work by Dr. Albert Hewett Coons, who pioneered antibody fluorescent labeling. The power of immunohistochemical analysis lies in its broad application, not only because it allows analysis across a broad range of tissues and target proteins, but for its utility across multiple disciplines. Over the decades, immunohistochemical staining has become an indispensable technique in basic research and at the clinic where it plays a crucial role in diagnosis and treatment.
This guide provides a comprehensive overview of immunohistochemistry techniques, addressing antibody basics such as antibody structure, function and specificity. Find answers to critical basic questions such as "What is a primary antibody", "What is the difference between antibody type and isotype", and "What are monoclonal and polyclonal antibodies?". Navigate the complexities of antibody selection, antibody conjugation, optimal experimental conditions, and sample preparation techniques. Become familiar with commonly used immunohistochemistry protocols, and instruments used for sample preparation and analysis.
Selecting and Handling antibodies
Antibodies are the central reagent in immunohistochemistry, but antibody selection is often complicated by the wide array of vendors with similar offerings. Learn valuable tips to choose the right antibodies among the wide selection and learn best practices for antibody handling and storage to ensure long lasting and reliable antibody performance.
Choosing antibody conjugation strategies
Selecting visualization strategies for optimal antigen detection may be challenging. Learn about the pros and cons of labeling primary or secondary antibodies to ensure specific and sensitive antigen detection. Additionally, learn to choose between fluorescent or enzyme reporters for antibody conjugation to meet experimental needs.
Achieving optimal experimental and sample conditions
Regardless of antibody specificity, successful immunostaining is dependent upon experimentation performed under optimal buffer and tissue conditions. The role of blocking, permeabilization, antibody dilution and antigen retrieval buffers, which optimize antibody binding and facilitate antigen detection, are discussed. In addition, methods for tissue preparation such as fixation, sectioning and storage are presented alongside images to introduce the beginner to instrumentation commonly used for frozen-tissue and paraffin-embedded tissue sectioning.
Immunohistochemistry protocols
Several basic immunohistochemistry protocols are presented with special emphasis on methodological rationale as well as advantages and caveats associated with each approach. Become familiar with the immunohistochemistry workflow. Learn about commonly used immunofluorescence and chromogenic-based detection methods including frequently used counterstaining tools for mapping antigens within tissues.
Overall, the more you know about the primary and secondary antibodies that you intend to use for immunohistochemistry, the more likely you are to achieve specific and consistent results. This guide provides a clear introduction to the wide range of reagents, methods and instruments needed for immunohistochemistry analysis. If you are new to the lab or new to immunohistochemical staining, this guide will provide a strong foundation to support your journey to becoming an expert.
Rosa Moreno, PhD.,
Product Marketing Specialist at Novus Biologicals LLC
