Immunostaining is a standard technique for the detection of specific proteins using antibodies. As the essential methods in numerous branches of scientific research, they have been widely used in various preclinical and clinical applications. These methods have been expanding and optimizing to further increase the scope of their use and improve their accuracy and reliability.
Types of Immunostaining
IHC is a variant of immunostaining and has been the most applied immunostaining technique. Before the straining process, the tissue or cells are preserved through fixation to maintain cell morphology and tissue architecture. To obtain high-quality immunostaining, it is necessary to overcome specific and non-specific backgrounds.
Flow Cytometry (FC)
The flow cytometer can be used for the quantitative analysis of stained cell products in the solution to determine the proportion and level of cells expressing specific markers. It is also possible to exclude dead cell material and analyze specific cell populations according to the size and granularity of the cells. Compared with IHC, FC presents improved sensitivity and can be analyzed in multiple colors to measure multiple antigens at the same time.
Western Blot (WB)
WB is a conventional molecular biology method that can be used for semi-quantitative analysis of expression levels of target proteins. The cells to be analyzed are lysed and the resulting proteins are electrophoresed on a gel to separate by size. Then the separated proteins are transferred to a membrane to be immunostained.
Enzyme-linked Immunosorption Assays (ELISA)
The ELISA is a more effective method for semi-quantitatively or quantitatively determining protein expression levels. Proteins in solution are adsorbed to ELISA plates through nonspecific binding or capture antibodies. The detection methods always include colorimetric and chemiluminescence.
Electron microscopy (EM) is suitable for the study of the detailed microarchitecture of cells or tissues. EM allows the detection of specific proteins in ultra-thin tissue sections and the subcellular localization of proteins
Fig.1 Micrograph of a GFAP immunostained section of a brain tumor.
Immunostaining in Neuroscience
Neurons are principal cells of the nervous system, and the neuronal diversity makes the cells study extremely challenging. To better understand the human nervous system, researchers focus on neural tissues, neural-specific protein targets, and neural cell markers. The identification of neuronal cells using immunostaining allows the study of signaling pathways and the elucidation of disease mechanisms. Subsequent two-dimensional imaging and 3D imaging can better capture neuronal features such as structure and dynamic direction.
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