Indirect Immunofluorescence of Tissues


Indirect immunofluorescence is a two‐step procedure whereby unlabelled antibodies are first bound to their target antigen and subsequently detected with fluorochrome‐conjugated secondary antibodies (indirect immunofluorescence). This technique is widely used for histological analysis of tissues in basic and clinical research.

Keywords: histology; fluorochrome; antibody; epifluorescence microscopy; confocal laser scanning microscopy

Figure 1.

Multiple immunofluorescence staining of neuronal markers in adult rat brain sections, as visualized in colour‐coded images from confocal laser scanning microscopy. (a) Concomitant visualization of neurons immunoreactive for the GABAA (γ‐aminobutyric acid type A) receptor α1 subunit (red) and axons positive for neuropeptide Y (green) in the olfactory bulb. Sections were incubated with a mixture of primary antibodies raised in guinea pig and rabbit, followed by a mixture of secondary antibodies (goat anti‐rabbit conjugated to Cy2 and goat anti‐guinea pig conjugated to Cy3). Images corresponding to the two fluorochromes were captured simultaneously and are displayed in a pseudo three‐dimensional rendering mode to illustrate that the two markers are fully segregated in this section. Bar, 10 μm.

(b) Triple immunofluorescence staining for the GABAA receptor α1 subunit (green; Cy2), the calcium‐binding protein parvalbumin (red; Cy3) and neuropeptide Y (blue; Cy5) in the CA1 region of the hippocampus. Note the pair‐wise, partial colocalization of these markers in subsets on neurons (arrowheads). Bar, 20 μm.

(c) Triple immunofluorescence staining for the calcium‐binding proteins parvalbumin (green; Cy2), calretinin (blue; Cy5) and neuropeptide Y (red; Cy3) in the cerebral cortex. Note that, unlike the hippocampus (b), the three markers are fully segregated in distinct subpopulations of neurons. For panels (b) and (c), signals corresponding to the three fluorochromes were captured simultaneously. The corresponding, colour‐coded images were superimposed from a stack of 12 confocal sections. Bar, 20 μm.



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Further Reading

Brelje TC, Wessendorf MW and Sorenson RL (1993) Multicolor laser scanning confocal immuno‐fluorescence microscopy: practical application and limitations. Methods in Cell Biology 38: 97–181.

Hanzel DK, Trojanowski JQ, Johnston RF and Loring JF (1999) High‐throughput quantitative histological analysis of Alzheimer's disease pathology using a confocal digital microscanner. Nature Biotechnology 17: 53–57.

Hockfield S, Carlson S, Evans C, Levitt P, Pintar J and Silberstein L (1993) Molecular Probes of the Nervous System, vol. I: Selected Methods for Antibody and Nucleic Acid Probes. New York: Cold Spring Harbor Laboratory Press.

Panchuk‐Voloshina N, Haugland RP, Bishop‐Stewart J et al. (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. Journal of Histochemistry and Cytochemistry 47: 1179–1188.

Sargent PB (1994) Double‐label immunofluorescence with the laser scanning confocal microscope using cyanine dyes. Neuroimage 1: 288–295.

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Härtig, Wolfgang, and Fritschy, Jean‐Marc(Apr 2001) Indirect Immunofluorescence of Tissues. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0002628]