This protocol is more complex and time consuming than the primary or direct protocol above, but it allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody. Immunofluorescence Immunofluorescence IF is a common laboratory technique, which is based on the use of specific antibodies which have been chemically conjugated to fluorescent dyes. Types of immunofluorescence There are two classes of immunofluorescence techniques, primary or direct and secondary or indirect.
Primary direct Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to a fluorophore. Secondary indirect Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first primary antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognises the primary antibody and binds to it.
Direct Indirect Time. See resources and tools for immuno-oncology research. Get resources and offers direct to your inbox Sign up. The fact that you have to use a conjugated secondary antibody to detect the primary antibody results in additional steps.
Secondary antibodies are relatively inexpensive compared to primary antibodies. Further cost savings may be made by using the same conjugated secondary antibody to detect different primary antibodies. Added complexity in indirect methods may result from having to select the appropriate secondary antibody. This is particularly relevant in multiplex experiments where several secondary antibodies, each targeting a different species and conjugated to different dyes, are needed.
The signal obtained in direct methods may seem weak when compared to indirect methods as signal amplification provided by the use of secondary antibodies does not occur. Several secondary antibodies will bind to the primary antibody resulting in an amplified signal.
Both approaches allow for differently-labeled antigens to be visualized using antibodies attached to fluorophores with distinct emission spectra, meaning that they can be imaged using separate laser lines. Schematic of direct labeled primary antibodies vs indirect two antibodies, secondary labeled immunofluorescence.
Immunofluorescence is commonly used in molecular and cell biology labs as a robust and simple method to reliably localize molecules on a wide range of fixed cells or tissues. Similarly, immunofluorescence is highly informative when studying steady state or endogenous protein levels and localization — fluorescence proteins, on the other hand, can sometimes multimerize or affect the kinetics and expression levels of molecules.
The antibody localizes in a pair of lines, one to on either side of the Z-disk, separated by nm. While confocal microscopy is widely used, newer designs of super resolution microscopes, such as STED Stimulated Emission Depletion microscopy and others, allow for nanoscopy and are capable of much higher resolution.
The antibodies are offered as highly functional conjugates with bright emission spectra that match the principal output wavelengths of common fluorescence instrumentation. DyLight Conjugates exhibit higher fluorescence intensity and photostability than many other dye conjugates.
The antibodies are designed for primary antibody detection and multiplex. ATTO-TEC fluorochrome conjugates offer strong absorption high extinction coefficient , high fluorescence quantum yield, and superior high photostability. Cy2 , Cy3 , Cy5 are popular choices for fluorescent labeling in applications such as fluorescence microscopy, flow cytometry, and fluorescent immunoassays. CyDyes are excellent alternatives to most other fluorescent dyes as they are brighter and offer greater photostability.
Depending on the specific CyDye, they may also produce less background and may be less sensitive to pH. Fluorescein Isothiocyanate FITC is a small organic molecule that is typically excited by the nm line of an argon laser and emission is collected at nm.
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