Immunofluorescence Microscopy

Immunofluorescence Microscopy 


Immunofluorescence (IF) microscopy is a particularly robust and broadly applicable method generally used by researchers to assess both the localization and endogenous expression levels of proteins of interest. 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. The effective application of this method comprises several considerations, including the nature of the antigen, specificity and sensitivity of the primary antibody, properties of the fluorescent label, permeabilization and fixation technique of the sample, and fluorescence imaging of the cell. Although each protocol will require fine‐tuning depending on the cell type, the antibody, and the antigen, there are steps common to nearly all applications. For more information see our technical tips for successful IF microscopy.

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.

Types of Fluorescent Microscopes   ATTO-TEC Fluorescent Dye Conjugates   
Assay Formats for Immunofluorescence Microscopy   CyDye® Fluorescent Dye Conjugates   
Fluorescein Conjugates

Texas Red Conjugates 

Rhodamine Conjugates Phycoerythrocyanin Conjugates 
Dylight™ Fluorescent Dye Conjugates   Allphycocyanin Conjugates 
  AMCA Conjugates

Featured Antibodies for Immunofluorescence Microscopy




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 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.


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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.

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  3. 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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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.



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Dy Light Fluorecent Dye




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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).


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Attotec 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.


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Cy Dye Conjugated Antibodies


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Fluorescein Dye Conjugates


Fluorescein isothiocyanate (FITC) is a small organic molecule that is typically excited by the 488 nm line of an argon laser, and emission is collected at 530 nm. FITC is currently the most commonly-used fluorescent dye for FACS but has use in wide-ranging applications like fluorescence microscopy, FLOW and immunofluorescence-based assays such as Western blotting and ELISA.


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Rhodamine (TRITC) Dye Conjugates


Rhodamine is a family of related fluorescent chemical compounds (fluorone dyes). Rhodamine dyes are used widely in biotechnology applications such as fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy and ELISA.


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Texas Red Dye Conjugates

Texas Red (Sulforhodamine 101 acid chloride) is a bright red fluorescent dye, used in a variety of applications including histology, FACS, fluorescence microscopy, and IHC. Texas Red fluoresces at about 615 nm, and absorbs at 561 or 589 nm.


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Phycoerythrin (RPE) Dye Conjugates

R-phycoerythrin (R-PE) is a large deeply bright phycobiliprotein complex (Mw 250kDa) isolated from red algae. RPE displays extremely bright red-orange fluorescence with high quantum yields. The sensitivity of RPE conjugates is usually 5 - 10x superior than those of the corresponding fluorescein conjugates. RPE is excited at 488 to 561 nm, and absobs at 496, 546, and 565 nm and a fluorescence emission peak at 578 nm. RPE can be used in various fluorescence-based applications like FLOW, microarray assays, ELISA, and other applications that need high sensitivity but not photostability.    


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Allophycocyanin (APC) Dye Conjugates

Allophycocyanin (APC) is a large protein (Mw ~105kDa) from the light-harvesting phycobiliprotein family found in Cyanobacteria and red algae. APC absorbs at 650nm and emits at 662nm. APC is usually used in FLOW, microarray assays, ELISA, and other applications that need high sensitivity but not photostability.


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AMCA Conjugates


AMCA provides an excellent additional fluorochrome in multilabeling experiments because this dye has virtually no overlap with other traditionally employed fluorochromes. Rockland uses 7-amino-3-((((succinimidyl)oxy)carbonyl)methyl)-4-methylcoumarin-6-sulfonic acid obtained from Molecular Probes Inc. Use a standard UV filter set for photography. Certain films which are blue sensitive may further enhance the detection of fluorescent staining using this dye.



Product Name   Catalog # Applications   Size
Streptavidin AMCA Conjugated   S000-15   FLISA, IF   1 mg


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The Rockland Advantage:  Immunofluorescence Microscopy

Seeing is believing. You can literally see the quality of Rockland antibodies using a variety of techniques including immunofluorescence microscopy. Visual evidence of proper target localization and low background staining is immediately apparent when cells are screened after staining.

 Protect your experiments with Rockland antibodies. Compromise elsewhere.






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Localization of HDAC1 poster







Fluorescent Labels Used for Antibody Conjugation by Rockland

Fluorescent Labels for Antibody Conjugation



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