Immunofluorescence is a very sensitive and versatile method used to label specific molecular targets within cells and tissues through combined use of specific antibodies and chemical fluorescent tags. It is commonly performed on immobilised, preserved specimens to label target proteins and their subsequent detection by means of fluorescent microscopy or flow cytometry, for diagnostic and biomedical research purposes. Importantly, it enables measurements of signal within single cells and thus provides information on sample heterogeneity, which is frequently lost in other methods. Over the decades, the use of immunofluorescent staining with its numerous adaptations has allowed to visualise discrete and delicate inner workings of the cells and tissues and brought to light how they respond to changes in context of health and disease.
Key Concepts
Specific, primary antibody is used for detection and quantification of target antigen in cell type of interest.
Signal amplification is achieved by binding of multiple fluorescently labelled secondary antibodies.
Appropriate fixation and permeabilisation methods should be applied for optimal sample staining.
Detection of fluorescence is achieved most commonly by epifluorescent or confocal microscopy, or flow cytometry analysis.
Immunofluorescent staining protocols have been extensively adapted for more advanced purposes, such as proximity ligation assay, fluorescence
in situ
hybridisation and super‐resolution microscopy.