Immunocytochemistry (ICC) is often called immunofluorescence (IF) and is characterized by imaging primary cells or cell lines in culture. Immunohistochemistry (IHC) is simply the detection of antibodies in tissue sections, whether it be by chromogenic or fluorescent realization methods. BioLegend products designated as IHC-P indicates the antibody is useful in formalin-fixed paraffin-embedded (FFPE) sections and IHC-F indicates the antibody is only useful in tissue that has been fixed and frozen prior to sectioning. If there is only an IHC designation, check with the literature citations to determine the method of tissue preparation compatible with each reagent.

Chromogenic vs. Fluorescent Imaging Methods

Chromogenic detection methods are advantageous because a signal can be amplified simply by extending the amount of time and substrate in the reaction. Also, it does not require sophisticated instruments for detection, only a microscope with phase contrast. HRP detection can, however, be accompanied by endogenous background associated with cellular peroxidase activity, non-specific signal and is only typically used to image a single marker at a time.

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IHC staining of anti-DJ-1 antibody (clone A16125E) on FFPE normal (left) and Parkinson's disease (right) brain tissue.

Fluorescent detection allows visualization of multiple markers at a time, most commonly through the use of discrete excitation sources optimal for each fluorophore. Fluorescent detection introduces the opportunity for advanced imaging applications as well, like live-cell imaging, multiphoton imaging, super-resolution microscopy, FLIM and FRET, just to name a few. Sensitivity can be a limitation of fluorescence microscopy at certain wavelengths, especially reagents that emit in the range of 350-450 nm due to increased autofluorescence of the sample. However, improved signal-to-noise can be obtained through varying enzymatic and immunologic amplification techniques, the use of higher sensitivity instrumentation, and near-infrared emitting fluorophores.

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Human paraffin-embedded colon tissue slices were Alexa Fluor® 594 anti-human Galectin-9 (clone 9M1-3, red) and Alexa Fluor® 647 anti-Vimentin (clone O91D3, green) and DAPI (blue).

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