Visualizing biological samples using a fluorescent microscope and antibodies conjugated to fluorescent labels is a powerful technique to localize structures within tissues, cells or subcellular compartments. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. Immunofluorescence microscopy is a widely used example of immunostaining and is a form of immunohistochemistry based on the use of fluorophores to visualize the location of bound antibodies.

Immunofluorescence can be used on tissue sections, cultured cells or individual cells that are fixed by a variety of methods. Antibodies can be used in this method to analyze the distribution of proteins, glycoproteins and other antigen targets including small biological and non-biological molecules.

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Types of Fluorescent Microscopes

Immunofluoresence microscopy can be used in several microscope designs for analysis of immunofluorescence samples. The simplest is the epifluorescence microscope. While confocal microscope 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.

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Assay Formats for Immunofluorescence Microscopy

There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect).

1. Primary (direct)
   
   Primary, or direct, immunofluorescence uses a single antibody that is conjugated directly to a fluorescent dye. The antibody recognizes the target molecule, binds to it and the conjugated fluorescent dye can be detected by the microscope. This technique, which reduces the number of steps in the staining procedure and is therefore faster, can avoid issues with antibody cross-reactivity or non-specificity which can lead to increased background signal. However, this method lacks any signal amplification inherent for the indirect method and requires laborious efforts by the scientist to conjugate potential numerous different primary antibodies required.



2. Secondary (indirect)
   
   Secondary, or indirect, immunofluorescence uses two antibodies; a primary antibody which recognizes the target biomolecule and binds to it and a secondary antibody conjugated to a fluorescent dye, which recognizes and binds to the primary antibody and indirectly localizes the target for detection by the microscope. While this protocol is more complex than the direct method, it is more flexible with regard to experimental design, results in greater signal detection through amplification and is easier in that secondary antibody conjugates are commercially available.

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Fluorescent Labels Used for Antibody Conjugation by Rockland

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ATTO-TEC Fluorescent Dye Conjugates

Rockland conjugates a select group of secondary antibodies to a new generation of patented fluorescent markers from ATTO-TEC including ATTO 425, ATTO 488, ATTO 532, ATTO 550 , ATTO 594, ATTO 647N and ATTO 655. The antibodies are designed for primary antibody detection and multiplex, multi-color analysis.  ATTO-TEC fluorochrome conjugates offer strong absorption (high extinction coefficient), high fluorescence quantum yield and superior high photostability. These conjugates are ideal for various immunofluorescence based assays including immunofluorescence microscopy, FLISA, STED microscopy, fluorescent western blotting, time resolved spectroscopy and FRET (Fluorescence Resonance Energy Transfer) applications as well as single-molecule detection (SMD).

• ATTO Secondary Antibodiy Conjugates

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CyDye^® Fluorescent Dye Conjugates

CyDye^® conjugated antibodies (i.e. 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.

• CyDye^® conjugated secondary antibodies for indirect immunofluorescence microscopy
• CyDye^® conjugated avidin and streptavidin
• CyDye^® conjugated anti-biotin.

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DyLight™ Fluorescent Dye Conjugates

DyLight™ conjugated antibodies (i.e. DyLight 405, DyLight 488,DyLight 549, DyLight 649, DyLight 680 and DyLight 800) are high-performance fluorescent conjugates for use as secondary antibody assays such as fluorescence microscopy, flow cytometry, western blotting, ELISA, high-content screening, multiplex assaysand other array platforms.  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.

• DyLight™ conjugated primary antibodies for direct immunofluorescence microscopy
• DyLight™ conjugated secondary antibodies for indirect immunofluorescence microscopy
• DyLight™ conjugated epitope tag antibodies for indirect immunofluorescence microscopy
• DyLight™ conjugated avidin and streptavidin
• DyLight™ conjugated anti-biotin  

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IRDye^® Fluorescent Dye Conjugates

IRDye^® infrared conjugated secondary antibodies (i.e.IRDye800, IRDye800CW, IRDye700 Antibody Conjugates) are ofte ncross-adsorbed for multiplex detection and provide high specificity for assays such as fluorescence microscopy, flow cytometry, western blotting, ELISA, multiplex assays and other array platforms. These antibody conjugates are optimized for high sensitivity andexcellent signal-to-noise performance on LI-COR Odyssey^® imaging systems and other detection platforms.

• IRDye^® conjugated primary antibodies for direct immunofluorescence microscopy
• IRDye^® conjugated secondary antibodies for indirect immunofluorescence microscopy
• IRDye^® conjugated epitope tag antibodies for indirect immunofluorescence microscopy
• IRDye^® conjugated avidin and streptavidin
• IRDye^® conjugated anti-biotin

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Other Fluorescent Dye Conjugates

• FITC conjugated secondary antibodies for indirect immunofluorescence microscopy 
• Rhodamine conjugated secondary antibodies for indirect immunofluorescence microscopy
• Texas Red^® conjugated secondary antibodies for indirect immunofluorescence microscopy

Browse all our fluorescent labeled primary antibodies 

Browse all our fluorescent labeled secondary antibodies