Inmunofluorescencia

Chapter 10 | Immunofluorescence
J. Paul Robinson PhD, Jennifer Sturgis BS and George L. Kumar PhD

Immunofluorescence (IF) is a common laboratory technique used in almost all aspects of biology. this technique based on pioneering work by Coons and Kaplan (1, 2), and later by Mary osborne (3), has been widely used both in research and clinical diagnostics. applications include the evaluation ofcells in suspension, cultured cells, tissue, beads and microarrays for the detection of specific proteins. IF techniques can be used on both fresh and fixed samples. In IF techniques, antibodies are chemically conjugated to fluorescent dyes such as fluorescein isothiocyanate (FItC) or tetramethyl rhodamine isothiocyanate (tRItC). these labeled antibodies bind (directly or indirectly) to theantigen of interest which allows for antigen detection through fluorescence techniques. the fluorescence can then be quantified using a flow cytometer, array scanner or automated imaging instrument, or visualized using fluorescence or confocal microscopy. the two main methods of immunofluorescent labeling are direct and indirect. Less frequently used is direct immunofluorescence whereby the antibodyagainst the molecule of interest is chemically conjugated to a fluorescent dye. In indirect immunofluorescence, the antibody specific for the molecule of interest (called the primary antibody) is unlabeled, and a second anti-immunoglobulin antibody directed toward the constant portion of the first antibody (called the secondary antibody) is tagged with the fluorescent dye (Figure 1).

Advantagesof direct immunofluorescence include shorter sample staining times and simpler dual and triple labeling procedures. In cases where one has multiple antibodies raised in the same species, for example two mouse monoclonals, a direct labeling may be necessary. Disadvantages of direct immunofluorescence include lower signal, generally higher cost, less flexibility and difficulties with the labelingprocedure when commercially labeled direct conjugates are unavailable. Advantages of indirect immunofluorescence include greater sensitivity than direct immunofluorescence. there is amplification of the signal in indirect immunofluorescence because more than one secondary antibody can attach to each primary (see Figure 1). Commercially produced secondary antibodies are relatively inexpensive,available in an array of colors, and quality controlled. Disadvantages of indirect immunofluorescence include the potential for cross-reactivity and the need to find primary antibodies that are not raised in the same species or of different isotypes when performing multiple-labeling experiments. Samples with endogenous immunoglobulin may exhibit a high background.

Primary antibody

DirectImmunofluorescence Fluorochrome Indirect Immunofluorescence Secondary antibody
Figure 2. Cultured pulmonary artery endothelial cells stained for tubulin (red), actin (green) and DNA (blue). The dual immunofluorescence procedure used rabbit anti-actin IgG and mouse anti-alpha tubulin IgG as primary antibodies. The secondary antibodies used were Texas Red-conjugated goat, anti-rabbit IgG and FITC-conjugatedgoat, anti-mouse IgG. The sample was also stained with the DNA-specific dye Hoechst 33342. Scale bar is equal to 20 microns.

Figure 1. Schematic of direct and indirect immunofluorescence.

IHC StaInIng MetHodS, FIFtH edItIon

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Immunofluorescence

Principle of Fluorescence
Fluorescence and phosphorescence are both types of luminescence. When molecules with luminescent propertiesabsorb light, they emit light of a different wavelength. With fluorescence the emission of light occurs extremely rapidly after the absorption of excitation light, whereas with phosphorescence emission continues for milliseconds to minutes after the energy source has been removed. Fluorescent materials give off light because of their atomic structure. electrons are arranged in discrete energy...