Products, protocols, tips and tricks for all your IHC needs
In an immunohistochemistry (IHC) experiment a primary antibody binds specifically to a protein of interest present in a tissue. The antibody binding is then visualized by a detection system, which provides information about if and where the protein is present in the tissue. IHC is a common method in diagnostics to determine morphological abnormalities and the presence of biomarkers indicative of certain diseases such as cancer.
Since the first IHC experiments in the 1940s much progress has been made in terms of developing different and more and more sensitive detection systems. In the beginning, secondary antibodies conjugated to enzymatic labels were used for visualization nearly exclusively. These days kits, directly conjugated primary antibodies and fluorescent labels are increasing in popularity. This trend is due to researchers wanting to detect several antigens simultaneously in one tissue specimen (a technique known as multiplexing).
When designing multiplexing experiments special care has to be taken at the fluorochrome and chromogen-enzyme combination selection stage, as it is important that the stained cellular compartments can be clearly spectrally differentiated; for example, two chromogen-enzyme pairs yielding a brown staining should be avoided.
When using fluorescently labeled antibodies, fluorochromes with a high spectral overlap should be avoided. It is therefore recommended to check for spectral overlap with the help of a fluorescence spectrum viewer when deciding which fluorochromes to use.
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