Immunofluorescence Technique
Immunofluorescence (IF) 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. It 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.
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.
IF 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.
Assay Formats for Immunofluorescence
There are two classes of IF techniques: primary (or direct) and secondary (or indirect):
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 using a microscope. This technique reduces the number of steps in the staining procedure (making it faster) and helps 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.
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.
Fluorescent Labels
| Fluorescent Label | Color | Abs (nm) | Em (nm) | MW (daltons) |
| DyLight™ 405 | Blue | 400 | 420 | 793 |
| 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 |
| Fluorecein (FITC) | Green | 495 | 528 | 390 |
| ATTO 532 | Yellow Green | 532 | 553 | 1,081 |
| 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 | 1,389 |
| Allophycocyanin (APC) | 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 |
| 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 |
DyLight™ Fluorescent Dye Conjugates
DyLight™ conjugated antibodies are high-performance fluorescent conjugates for use as secondary antibody assays such as fluorescence microscopy, flow cytometry, western blotting, ELISA, high-content screening, multiplex assays, and 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.
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. 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 Fluorescence Resonance Energy Transfer (FRET) applications, as well as single-molecule detection (SMD).
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.
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.
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.
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, 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.
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.
AMCA Conjugates
AMCA provides an excellent additional fluorochrome in multi-labeling 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.