Immunofluorescence

Abstract

Immunofluorescence is a very sensitive and versatile method used to label specific molecular targets within cells and tissues through combined use of specific antibodies and chemical fluorescent tags. It is commonly performed on immobilised, preserved specimens to label target proteins and their subsequent detection by means of fluorescent microscopy or flow cytometry, for diagnostic and biomedical research purposes. Importantly, it enables measurements of signal within single cells and thus provides information on sample heterogeneity, which is frequently lost in other methods. Over the decades, the use of immunofluorescent staining with its numerous adaptations has allowed to visualise discrete and delicate inner workings of the cells and tissues and brought to light how they respond to changes in context of health and disease.

Key Concepts

  • Specific, primary antibody is used for detection and quantification of target antigen in cell type of interest.
  • Signal amplification is achieved by binding of multiple fluorescently labelled secondary antibodies.
  • Appropriate fixation and permeabilisation methods should be applied for optimal sample staining.
  • Detection of fluorescence is achieved most commonly by epifluorescent or confocal microscopy, or flow cytometry analysis.
  • Immunofluorescent staining protocols have been extensively adapted for more advanced purposes, such as proximity ligation assay, fluorescence in situ hybridisation and superÔÇÉresolution microscopy.

Keywords: immunofluorescence; antibody; antigen; fluorophore; cell fixation; confocal microscopy; FACS

Figure 1. Direct vs indirect immunofluorescence. (a) Direct immunofluorescence. The primary antibody specific to the antigen of interest is directly conjugated with a fluorescent, chemical dye. (b) Indirect immunofluorescence. Specific primary antibody bound to the target is detected by a secondary antibody, raised against the host species of the primary antibody. Multiple labelled secondary antibodies can bind to a single primary antibody leading to a signal amplification.
Figure 2. The process of immunofluorescent staining of cells grown in culture. Cells grown in in vitro culture are seeded onto a glass coverslip and when appropriate, briefly washed with phosphate‐buffered saline (PBS) and fixed using aldehyde fixation or dehydration with organic alcohols. Once the biomolecules are cross‐linked, it is possible to permeabilise the sample to allow antibody to enter the cell (if the target epitope is located within the cell). Blocking buffer is then used to mask any unspecific binding and to reduce unspecific staining before the primary antibody is applied. Following incubation with primary antibody the excess is washed away with saline and sample is incubated with a labelled, secondary antibody. A nuclear counterstain such as DAPI (usually emitting blue light) can also be used at this stage. Excess antibody is washed away and the specimen is preserved in mounting medium and stored in the dark until imaging using a fluorescent microscope.
Figure 3. The physics of fluorescence. (a) Simplified Jablonski diagram depicting the transition of an electron from a ground (nonexcited) state S0, to an excited singlet states S1 and S2 due an absorption of light. The energy of the electron is then dissipated through nonradiative transition in form of heat and vibrations. Finally, a portion of electrons returns to ground state emitting light with a longer wavelength than the excitation light. (b) Diagram depicting the Stokes shift principle where the maximum peak of the excitation light is distinct from the peak of the emitted, more red‐shifted, light.
Figure 4. Detection methods. (a) Schematic diagram of an optical set up used in confocal microscopy. The light from a laser source is concentrated by optical lenses and reflected by a dichroic mirror to subsequently pass through an objective. It is then focused to form a cone of light with defined size and shape that illuminates the sample forming a confocal volume. The emitted light is directed back, filtered and further focused by passing through a pinhole down to detectors. The laser scans the selected area at a point by point, line by line basis. (b) Diagram representing an optical set up of an epifluorescent microscope. Fluorescent light generated by an arc lamp is concentrated by a set of optical lenses and illuminates a whole sample plane. The signal is then emitted back and filtered to cut off nonspecific light into a sensitive and fast‐recording camera. (c) Diagram representing setup of a flow cytometer. Cell suspension is directed in a single file to pass through an excitation light. The intensity of emitted light and light scatter from each single cell that passes the detector is then analysed.
Figure 5. Example image obtained by indirect immunofluorescent staining. Image of mixed neuronal/astrocyte cultures in vitro. The mouse primary antibody against astrocyte marker GFAP was stained with anti‐mouse secondary antibody conjugated with Alexa‐555 (red). Antibody against neuronal marker β‐tubulin‐III in red stained with Alexa‐488 (green). Nucleus was counterstained with DAPI (in blue). Image taken using confocal Zeiss 7.80 microscope, scale bar represents 10 µm.
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Further Reading

Alivisatos AP, Gu W and Larabell C (2005) Quantum dots as cellular probes. Annual Review of Biomedical Engineering 7 (1): 55–76.

Lichtman JW and Conchello JA (2005) Fluorescence microscopy. Nature Methods 2 (12): 910–919.

Odell ID and Cook D (2013)Immunofluorescence Techniques 133 (1): e4.

Shakes DC, Miller DM and Nonet ML (2012) Immunofluorescence microscopy. Methods in Cell Biology 107: 35–66.

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Wardyn, Joanna D, and Jeyasekharan, Anand D(Oct 2018) Immunofluorescence. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001174.pub2]