Immunofluorescence (IF) is a technique that uses antibodies and fluorescence imaging to visualize target proteins and other biomolecules in fixed cells or tissue specimens.
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This method can disclose the location, relative expression, and even activation states of target proteins. The value of IF lies in its ability to deliver data that is both graphical and quantitative.
In IF investigations, proteins of interest can be identified using either primary antibodies covalently attached to fluorophores (direct detection) or a two-step technique using an unlabeled primary antibody followed by a fluorophore-linked secondary antibody (indirect detection).
Either method lets the user combine numerous fluorophores (multiplex analysis), making IF suitable for studying protein co-localization, changes in subcellular localization, differential activation of proteins inside a cell, identifying various cell subsets, and other studies.
The goal of Cell Signaling Technology (CST) is to generate highly specific antibodies that produce a strong, specific signal with a low background. CST’s scientists screen a vast number of antibodies and only recommend those that are most appropriate for the application.
CST’s validation efforts involve extensive protocol optimization and antibody titration to establish the optimal working conditions for each antibody. Its scientists also validate supporting reagents, such as fluorophore-linked secondary antibodies, to improve antigen detection and IF protocol efficiency.
This eBook highlights the crucial steps in CST’s protocol for IF, introduces key concepts related to antibody performance and design of controls, and offers supporting data to explain CST’s recommendations.
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