Immunohistochemistry (IHC) experiments are used to visualize antigens/proteins in a tissue with the help of enzymatically or fluorescently labeled antibodies. The technique is commonly applied in the clinic and in academic research to distinguish healthy from morphologically abnormal tissue. Depending on how the tissue has been prepared, the IHC technique is referred to as IHC-paraffin, IHC-frozen or IHC-free floating.
As with other antibody based applications, in recent years, a trend towards multiplexing (simultaneous visualization of several protein antigens) has been observed.
When designing multiplexing experiments special care has to be taken at the fluorophore 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, fluorophore 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 fluorophore to use.
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