What is Fluorescein (FITC)?
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used extensively in
fluorescence microscopy and
immunohistochemistry. It is a small organic compound that exhibits a bright green fluorescence when excited by ultraviolet or blue light. FITC is widely used to label antibodies and other molecules in histological studies, allowing researchers to visualize specific proteins or structures within
tissues.
How is FITC Used in Histology?
In the context of histology, FITC is primarily used in
fluorescence microscopy techniques. It is conjugated to
antibodies that are specific to the target antigen. Once the FITC-labeled antibody binds to its target on the tissue section, the sample can be observed under a fluorescence microscope. This allows for the detection and localization of specific proteins within the tissue, providing valuable information about their distribution and abundance.
Sensitivity: FITC provides high sensitivity, allowing for the detection of
low-abundance proteins within tissues.
Visualization: The bright fluorescence of FITC makes it easy to visualize labeled structures against the background of the tissue.
Versatility: FITC can be conjugated to a wide range of molecules, including proteins, peptides, and nucleic acids, making it highly adaptable for different experimental needs.
Photobleaching: FITC is prone to
photobleaching, where prolonged exposure to light causes the fluorescence to diminish over time.
Overlapping Spectra: FITC's emission spectrum can overlap with other fluorophores, potentially complicating
multicolor experiments.
Poor Stability: FITC may not be the best choice for experiments requiring long-term storage of labeled samples as it can degrade over time.
Activation: The protein of interest is activated, often through the introduction of amine groups.
Reaction: The activated protein is mixed with FITC under controlled conditions, allowing the isothiocyanate group of FITC to form a stable covalent bond with the protein.
Purification: The FITC-conjugated protein is purified to remove any unbound FITC, ensuring specificity in subsequent applications.
Light Exposure: Minimize exposure to light to prevent photobleaching before and during experiments.
pH Sensitivity: Maintain optimal pH levels during storage and experiments, as FITC's fluorescence is pH-sensitive.
Storage Conditions: Store FITC-labeled compounds in the dark at low temperatures to preserve their fluorescence activity.
Conclusion
Fluorescein (FITC) remains a cornerstone in histological studies due to its sensitivity and versatility. While it has some limitations, careful handling and alternative options can mitigate these issues, allowing FITC to continue playing a vital role in the visualization and analysis of biological tissues.
