[Practical Basis of Immunohistochemical Research and Multiplexed Immunohistochemistry]

Immunohistochemistry(IHC)is a central tool of modern morphology. IHC is widely used from basic research to diagnostic practice utilizing its advantage in visualizing ability of in situ distribution of target antigens. In order to perform proper immunohistochemical analyses, it is necessary to fully understand the principle of IHC to prepare appropriate samples, to select suitable antibodies, and to apply reasonable method. The final output of IHC and conventional multi-labeled IHC, are given as histological images. Interpretation of those images is entrusted to the researcher. Therefore, reproducibility and verifiability of those subjective explanation of IHC images are occasionally insufficient as compared to other experimental methods such as(multi-colored)flow cytometry. In recent years, extremely multiplexed IHC method(multiplex immunohistochemistry: mIHC)by various methodological approach have been developed and put into practical use. By mIHC, morphological factors, namely coordinate of cells and expression level of target antigens, are established as matrices of numeral values, and mathematical analyses of tissue morphology is now practical. In this essay, basic matters to keep in mind at performing proper IHC are reconfirmed, and present a methodology of multiplexed IHC including mIHC. In addition to their features and advantages, unsolved issues of mIHC are also taken up.