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Evolution and Future of Multiplex Immunohistochemistry using Tyramide Signal Amplification - Part 4 of 4Jane Naberhuis, Ph.D.ArticleSince fluorescent labels were first conjugated to antibodies against targets of interest in the 1940s, a number of landmark developments have enabled the development of immunohistochemistry. These developments include antibody purification and labeling, enzyme digestion and heat-induced approaches to epitope retrieval, signal amplification, and improvements in imaging technologies.
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Benefits of Fluorescent Multiplex Immunohistochemistry using Tyramide Signal Amplification - Part 3 of 4Jane Naberhuis, Ph.D.ArticleFluorescent multiplex immunohistochemistry (mIHC) with tyramide signal amplification (TSA) has several advantages over one-color or traditional mIHC.
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Practical Overview of Multiplex Immunohistochemistry using TSA - Part 2 of 4Jane Naberhuis, Ph.D.ArticleSimultaneous detection of multiple distinct proteins of interest within a single sample can be achieved with carefully optimized fluorescent multiplex immunohistochemistry (mIHC) using tyramide signal amplification (TSA). This technique utilizes an unconjugated primary antibody specific to the protein of interest, and a primary specific secondary antibody conjugated to horseradish peroxidase (HRP). Detection is achieved with the fluorophore-conjugated HRP substrate, tyramide. When activated, tyramide forms covalent bonds with the tyrosine residues on or near the protein of interest. This permanent deposition allows the primary/secondary antibody pair to be stripped from the sample without disrupting the antigen-associated fluorescence signal. Thus, multiple rounds of staining can be performed in sequence on a given sample, without fear of the crosstalk that would otherwise result from using multiple primary antibodies raised in a single species.
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An Introduction to Multiplex Immunohistochemistry - Part 1 of 4Jane Naberhuis, Ph.D.ArticleWith the advancement of immunotherapeutics, the urge to understand the tumor microenvironment has never been more pressing. Immunohistochemistry is a powerful tool used to examine protein expression, distribution, and activation in situ. Antibodies specific to an antigen of interest are used to detect the antigen in thin sections of flash frozen or formalin-fixed paraffin-embedded tissue. Visualization of the antigen is achieved using either an enzymatic reaction that induces chromogen precipitation at the site of antibody-antigen binding, or fluorescent reporters. Fluorescent reporters may be directly conjugated to the primary antibody used to detect the antigen of interest (direct immunofluorescence), or may be attached to a secondary antibody that detects the species-specific primary antibody (indirect immunofluorescence). The latter is more common, as it achieves more sensitive antigen detection.
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