Multiplexing And Spectral Microscopy

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Book Chapter

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Methods in Molecular Biology


Visualization of proteins within a tissue sample via immunohistochemistry (IHC) is a central aspect of pathology. However, such methods are limited to the detection of one or two proteins, due to the overlapping absorption/emission spectra of chromogens and fluorescent dyes. The advent of spectral microscopy has enabled improved visualization of multiple proteins by allowing for the specific light wavelengths/spectral signatures of individual fluorophores and chromogens to be unmixed and analyzed, thus detecting signals that would be indistinguishable with conventional microscopy. Combined with improvements to multiplexed immunohistochemistry (mIHC) protocols, spectral microscopy facilitates the interrogation of spatial relationships between four (enzymatic mIHC) or seven (fluorescent mIHC) proteins, unlocking the wealth of information contained within a single tissue section. Furthermore, the application of linear unmixing for image analysis allows for a reduction in background signal associated with tissue autofluorescence and can distinguish chromogens with similar absorption spectra to identify protein colocalization in brightfield spectral microscopy. While many mIHC protocols have been optimized for spectral microscopy, this chapter will focus in detail on two common methods: enzymatic mIHC and manual fluorescent mIHC using tyramide signal amplification and microwave technology.

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Immunohistochemistry and Immunocytochemistry Methods and Protocols