Enzymatic or fluorescence signals given by conjugated antibodies are subsequently coupled to bright-field or fluorescence microscopy, respectively. The suffixes chemistry and fluorescence are also assigned when enzymes or fluorochromes are used for antibody labeling ( Table 1).
Technically, to identify the direction in which this procedure will be applied, the addition of prefixes, such as cyto or histo, is used when this method describes antigenic sites within cells or tissues, respectively. In addition to being used as a routine laboratory work-up, the development of immunolocalization protocols itself is an area of deep research and upgrades. Remarkably, to date this method has prevailed, essentially preserving its initial methodological design.
Immunostaining, also known as immunolabeling or immunolocalization, is one of the most widely used techniques in clinical diagnosis, and in both biological and histopathological research this method is applied to describe the localization of protein populations, distributions, as well as abundance. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles.
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