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Immunofluorescence Microscopy


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.  


Background 

ATTO-TEC Fluorescent Dye Conjugates  

Types of Fluorescent Microscopes 

List of Fluorescent Labels Used for Antibody Conjugation by Rockland  

Assay Formats for Immunofluorescence Microscopy 

CyDye® Fluorescent Dye Conjugates  

List of Fluorescent Labels Used for Antibody Conjugation by Rockland 

IRDye® Fluorescent Dye Conjugates 

Dylight™ Fluorescent Dye Conjugates 

Other Fluorescent Dye Conjugates 




microscopy-1 background

Background

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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microscopy-2 types

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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microscopy-3 types

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

 

Fluorescent label

 

 

Color

 

 

Abs (nm)

 

 

Em (nm)

 

 

MW (daltons)

 

DyLight™ 405

Violet

400

420

793

Aminomethylcoumarin (AMCA)

Violet Blue

353

442

410

ATTO 425

Blue

436

484

498

Cy2™

Blue Green

489

505

897

DyLight™ 488

Blue Green

493

518

1,011

ATTO 488

Green

501

523

981

Fluorescein (FITC)

Green

495

528

390

ATTO 532

Yellow Green

532

553

1081

Cy3™

Yellow Green

552

565

949

DyLight™ 549

Yellow Green

550

568

982

Rhodamine (TRITC)

Orange

550

570

444

R-Phycoerythrin (RPE)

Orange

488

575

240,000

ATTO 550

Orange

554

576

791

Cy3.5™

Orange Red

581

596

1,286

Texas Red®

Red

596

620

625

ATTO 594

Red

601

627

1389

Allophycocyanin

Far-Red

650

660

100,000

Cy5™

Far-Red

650

667

975

ATTO 647N

Far-Red

644

669

843

DyLight™ 649

Far-Red

646

674

1,008

ATTO 655

Far-Red

663

684

887

IRDye®700DX

Near Infra-Red

689

700

1,954

Cy5.5™

Near Infra-Red

678

703

1,312

DyLight™ 680

Near Infra-Red

682

715

950

DyLight™ 800

Infra-Red

770

794

1,050

IRDye® 800CW 

Infra-Red

774

800

1,166

IRDye® 800

Infra-Red

778

806

1,166

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microscopy-5 attotec

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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microscopy-6 cydye

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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microscopy-7 dylight

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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microscopy-8 IRDye

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.

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microscopy-9 other

Other Fluorescent Dye Conjugates

Browse all our fluorescent labeled primary antibodies 

Browse all our fluorescent labeled secondary antibodies 

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