Fluorescence imaging–based techniques are highly useful for interrogating the structural and functional of aspects of cells and tissues. When combined with immunochemistry, their analytical power increases tremendously, enhancing their utility in a wide range of research and clinical applications, making them invaluable tools. Immunofluorescence microscopy revolutionized the field of cell biology by enabling live-cell imaging to visualize whole organelles. Immunohistochemistry and immunocytochemistry are common fluorescence-imaging techniques that combine the power of antigen-antibody binding for cell analysis.
What is immunofluorescence and what are its applications?
Immunofluorescence (IF) is a powerful immunostaining technique that utilizes microscopy to visualize fluorophore-conjugated antibodies bound to target proteins and other molecules of interest. IF is used to identify cell- and tissue-specific antigens in cells; visualize the presence or absence, cellular localization, and activation status of proteins; and analyze immune responses.
Principles and types of immunofluorescence
IF exploits the property of fluorescent molecules or fluorophores to absorb photons at a certain wavelength (absorption spectrum of the molecule) and emit them at a higher wavelength after a brief interval (emission spectrum) with an accompanied energy loss. The emitted fluorescence can be visualized by microscopy. Fluorophores can be excited by visible or UV light.
Fluorescent dyes with high photostability and fluorescence quantum yield are commercially available with excitation maxima spanning a range of wavelengths, from 400 to >700 nm. They do not damage living cells and can be safely used in biological preparations.
For immunofluorescence, cells or tissues are first fixed and permeabilized. For immunostaining, the fluorophores are conjugated to antibodies against antigens of interest and the fluorescence signal is then visualized using imaging microscopy. IF can be grouped into two types—direct and indirect—based on the antibody used and signal amplification needed.
Direct immunofluorescence
A single antibody (the primary antibody) is used for immunostaining and detecting the protein of interest. The fluorophore-conjugated primary antibody binds directly to the antigen of interest and is visualized using imaging microscopy.
Advantages of direct IF
- Decreased species cross-reactivity issues as the need for choosing different species reactivity for two antibodies is eliminated
- Decreased time (fewer steps) compared to indirect IF
Disadvantages of direct IF
- Does not allow for signal amplification through a secondary antibody
- Decreased sensitivity of detection
- Limited choice of fluorophore-conjugated primary antibodies
- More expensive compared to detection using fluorescent secondary antibodies
Indirect immunofluorescence
Two antibodies (a primary and a secondary antibody) are used for immunostaining and detecting the protein of interest. First, the protein of interest is labeled with a specific primary antibody. A fluorophore-conjugated secondary antibody (with a different species reactivity than the primary antibody) then recognizes the bound antigen-antibody complex and binds to the primary antibody. Since more than one secondary antibody can bind to the primary antibody, the fluorescence signal is amplified, providing more sensitivity of detection.
Advantages of indirect IF
- Signal amplification through number of secondary antibodies being able to bind to the primary antibody
- Increased sensitivity of detection through signal amplification compared to direct IF
- Wide array of choices for fluorescent-labeled secondary antibodies
- Less expensive compared to detection using fluorophore-conjugated primary antibodies
Disadvantages of indirect IF
- Increased species cross-reactivity issues as two antibodies with two different species reactivity are required
- Increased time (more steps) compared to direct IF
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