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Overview
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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. 

 
Relative absorption and emission spectra.
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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.

 
Diagram showing direct immunofluorescence.
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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
 
Diagram showing direct immunofluorescence.
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IF Microscopy, ICC and IHC
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What is immunofluorescence microscopy and what are its applications?

Both direct and indirect IF utilize imaging microscopy for visualization of fluorescence signals. Depending on the experimental need and requirement, epifluorescence or confocal microscopy is used for imaging.

 

Epifluorescence or wide-field microscopy

Allows visualization of cellular morphology, presence and localization of cellular proteins, and markers and analysis of cellular phenotypes. 

 

Confocal microscopy

Used when a 3-D image of the sample with high resolution is required. Used to analyze subcellular localization of proteins, colocalizations and protein-protein interactions.

 

Typical fluorescence microscopy channels

Various wavelength results for immunofluorescence.

Immunocytochemistry, immunohistochemistry and immunofluorescence—what are the differences?

Both immunocytochemistry (ICC) and immunohistochemistry (IHC) employ immune labeling of antigens for analysis and use immunofluorescence methods. Sometimes, immunocytochemistry and immunofluorescence are used interchangeably.

 

While ICC focuses on analysis at a cellular level, IHC allows examination of the whole tissue. Both techniques use immunofluorescence for detection. It is also possible to use enzyme-based detection (e.g., using 3,3’-diaminobenzidine (DAB)).

 

 
Wavelength results for BV421, BV480, 488 Fluor, 568 fluor, 647 fluor dyes.
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Robust and easy 5-color imaging

Advantages of immunofluorescence-based detection over enzymatic detection
  • Ability to quantitate fluorescence signals (as opposed to qualitative determination using enzyme-based methods)
  • Ability to multiplex (fluorescent dyes with different emission spectra can be combined to detect multiple proteins)
  • Photostability of fluorescent dyes

 

Photostability of BV dyes

Right image: Continuous exposure to excitation light. Cells: MCF7 cells. Fixation: BD Cytofix™ Fixation Buffer (Cat. No. 554655). Antibodies: Purified Mouse Anti-Human CD324 (Cat No. 562869) with BD Horizon Brilliant Violet™ 480 (BV480) Goat Anti-Mouse (Cat. No.564877), BD Horizon Brilliant Violet™ 421 (BV421) Goat-Anti-Mouse (Cat. No. 563846), or Alexa Fluor™ 488 Goat Anti-Mouse (Thermo Fisher Scientific). Other reagents: ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific). Instrument: ImageXpress™ Micro XLS System (Molecular Devices). 20X objective.

 
Chart showing fluorescence intensity over time for BV421, BV480, Alexa Fluor® 488.
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Recommended Filter Configurations
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Determining your optical configuration while adopting the BD Horizon Brilliant™ Fluorophores
Depending on the type of microscopy used and other requirements, you can obtain a recommended filter configuration.
  • ChannelDyesExcitation (nm)DC (nm)Emission (nm)Optimized Filter Sets
    BlueDAPI377/50409430/24 or 440/40Chroma or Semrock
    AquaBD Horizon Brilliant
    Violet™ 480 (BV480)
    445/20 or 438/24458483/32Chroma or Semrock
    GreenFITC, Alexa Fluor™ 488,
    eGFP, zsGreen
    490/30 or 485/20506 or 495537/26 or 525/50Chroma or Semrock
    OrangeAlexa Fluor™ 555,
    Alexa Fluor™ 568
    Any optimized filter setAny optimized filter setAny optimized filter setAny vendor
    RedAlexa Fluor™ 594Any optimized filter setAny optimized filter setAny optimized filter setAny vendor
    Far RedAlexa Fluor™ 647Any optimized filter setAny optimized filter setAny optimized filter setAny vendor

    Recommended filter configuration to adopt BD Horizon Brilliant Violet™ 480 (BV480) for improved immunofluorescence assays.

  • Configuration for one brighter dye: BD Horizon Brilliant Violet™ 421 (BV421) for improved immunofluorescence assays.

     

    ChannelDyesExcitation (nm)DC (nm)Emission (nm)Optimized Filter Sets
    BlueBD Horizon Brilliant
    Violet™ 421 (BV421)
    392/23 or 377/50409430/24 or 440/40Chroma or Semrock
    AquaBD Horizon Brilliant
    Violet™ 480 (BV480)
    438/24 or 445/20458483/32Chroma or Semrock
    GreenFITC, Alexa Fluor™ 488,
    eGFP, zsGreen
    490/30 or 485/20506 or 495537/26 or 525/50Chroma or Semrock
    OrangeAlexa Fluor™ 555,
    Alexa Fluor™ 568
    Any optimized filter setAny optimized filter setAny optimized filter setAny vendor
    RedAlexa Fluor™ 594Any optimized filter setAny optimized filter setAny optimized filter setAny vendor
    Far RedAlexa Fluor™ 647,
    DRAQ5™ (DNA)
    Any optimized filter setAny optimized filter setAny optimized filter setAny vendor

    Recommended filter configuration to adopt BD Horizon Brilliant Violet™ 421 (BV421) for improved immunofluorescence assays.

     

     

    Configuration for two brighter dyes: BD Horizon Brilliant Violet™ 421 (BV421) and BV480 for
    improved immunofluorescence assays.

     

     

    ChannelDyesExcitation (nm)DC (nm)Emission (nm)Optimized Filter Sets
    BlueBD Horizon Brilliant
    Violet™ 421 (BV421)
    392/23 or 377/50409430/24 or 440/40Chroma or Semrock
    AquaBD Horizon Brilliant
    Violet™ 480 (BV480)
    438/24 or 445/20458483/32Chroma or Semrock
    GreenFITC, Alexa Fluor™ 488,
    eGFP, zsGreen
    490/30 or 485/20506 or 495537/26 or 525/50Chroma or Semrock
    OrangeAlexa Fluor™ 555,
    Alexa Fluor™ 568
    Any optimized filter setAny optimized filter setAny optimized filter setAny vendor
    RedAlexa Fluor™ 594Any optimized filter setAny optimized filter setAny optimized filter setAny vendor
    Far RedAlexa Fluor™ 647,
    DRAQ5™ (DNA)
    Any optimized filter setAny optimized filter setAny optimized filter setAny vendor
  • Up to 5-Color ConfigurationLaser LineDyes
    DAPI Essential405 nmDAPI
    440 nm or 458 nmBD Horizon Brilliant Violet™ 480 (BV480)
    488 nmAlexa Fluor™ 488 or GFP
    561 nmAlexa Fluor™ 568 or RFP
    633 nmAlexa Fluor™ 647
    Alternate DNA Stain405 nmBD Horizon Brilliant Violet™ 421 (BV421) and
    BD Horizon Brilliant Violet™ 480 (BV480)
    440 nm or 458 nmLaser line not essential
    488 nmAlexa Fluor™ 488 or GFP
    561 nmAlexa Fluor™ 568 or RFP
    633 nmDRAQ5™ or Alexa Fluor™ 647
Sample Data
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Sample data generated using BD Biosciences antibodies and reagents

Multicolor fluorescence immunohistochemistry staining of mouse spleen tissue

Spleen cryosections (5 µm) from mice expressing CX3CR1-green fluorescent protein (pseudo-colored green) and CCR2-red fluorescent protein (pseudo-colored red) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), blocked with 5% goat serum and 1% BSA diluted in 1X PBS, and stained with BD Horizon Brilliant Violet™ 421 (BV421) Rat Anti-Mouse CD4 (Cat. No. 562891) (pseudo-colored magenta), Alexa Fluor™ 647 Rat Anti-Mouse CD45R/B220 (Cat No. 557683) (pseudo-colored yellow), and BD Horizon Brilliant Violet™ 480 (BV480) Rat Anti-Mouse CD3 Molecular Complex (pseudo-colored cyan). Slides were mounted with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific) and the image was captured on a BD Pathway 435 Cell Analyzer (epifluorescence microscope) and merged using BD Attovision Software. 20X objective. Carried out in collaboration with the Catherine Hedrick lab at La Jolla Institute of Allergy and Immunology.

 
Grid of 6 fluorescence results from mouse spleen sample.
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Multicolor immunocytochemistry using BD Horizon™ BV480

MCF-7 cells (ATCC, HTB-22) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100 and blocked with 5% goat serum, 1% BSA, 0.5% Triton™ X-100 in 1X PBS. Cells were incubated with Purified Mouse Anti-E-Cadherin (Cat. No. 610182) and the second-step reagent was BD Horizon Brilliant Violet™ 480 (BV480) Goat Anti-Mouse (Cat. No. 564877) (pseudo-colored aqua). Cells were then stained with directly conjugated antibodies: Alexa Fluor™ 488 Mouse Anti-β-Tubulin (Cat No. 558605) (pseudo-colored green), Alexa Fluor™ 555 Mouse Anti-Human Ki-67 (Cat. No. 558617) (pseudo-colored purple), Alexa Fluor™ 647 Mouse Anti-GM130 (Cat. No. 558712) (pseudo-colored red). DAPI (Cat. No. 564907) was used as a nuclear counterstain (pseudo-colored blue). Slides were mounted with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific) and the image was captured on a BD Pathway 435 Cell Analyzer and merged using BD Attovision Software (BD Biosciences). 20X objective.

 
Grid of 6 fluorescence results for BV480
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Multicolor immunofluorescence staining of human embryonic stem cells

Cultured cells from the H9 human ES cell line were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100 diluted in 1X PBS, blocked with 5% goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1X PBS, and stained with BD Horizon Brilliant Violet™ 421 (BV421) Mouse Anti-SSEA-1 (Cat. No. 562705), BD Horizon Brilliant Violet™ 480 (BV480) Mouse Anti-CD324(E-Cadherin) (Cat. No. 565646), Alexa Fluor™ 488 Mouse Anti-Oct3/4 (Cat. No. 561628), and Alexa Fluor™ 555 Mouse Anti-Human Ki-67 (Cat No. 558617). DRAQ5™ (Cat. No. 564902) was used as a nuclear counterstain. Slides were mounted with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific) and the images were captured on a BD Pathway 435 Cell Analyzer (epifluorescence microscope) and merged using BD Attovision Software. 20X objective.

 
Various fluorescence results for multiple tests.
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Compatibility of BD Horizon™ BV480 with BD Horizon™ BV421 for immunofluorescence imaging

Cells: H9 human embryonic stem cells (WiCell, Madison WI). Fix: BD Cytofix™ Fixation Buffer (Cat. No. 554655); Perm: BD Phosflow™ Perm Buffer III (Cat. No. 558050), Block/Stain: 5% goat serum, 1% BSA, 0.5 % Triton™ X-100 in 1X PBS. Antibodies: Purified Mouse Anti-Oct3/4a (Cat. No. 561555) with BD Horizon Brilliant Violet™ 480 (BV480) Goat Anti-Mouse (Cat. No. 564877), BD Horizon Brilliant Violet™ 421 (BV421) Mouse Anti-Human TRA-1-60 Antigen (Cat. No. 562711). Other reagents: DRAQ5™ (BioStatus), ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific). Instrument: BD Pathway 435 Cell Analyzer and images merged using BD Attovision Software (BD Biosciences). 20X objective.

 
Spectra for BV480 results.
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Alexa Fluor and ProLong are trademarks of Thermo Fisher Scientific.

DRAQ5 is a trademark of BioStatus Limited. ImageXpress is a trademark of Molecular Devices.

Triton is a trademark of Dow.