The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.
Immunofluorescence is a technique that allows the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections. Immunofluorescence stained samples are examined under a fluorescence microscope or confocal microscope.
Because fluorescent dyes such as fluorescein and rhodamine can be coupled to antibodies without destroying their specificity, the conjugates can complex with antigen and be visualized via fluorescence microscopy. The microscope excites the chosen dyes by light of one or more wavelengths, which in turn emits light at a characteristic wavelength captured by a selective filter. The image is then projected with localized regions of fluorescence indicating different antigens labeled by antibodies of distinctive color.
| Common Fluorescent Tags | Excitation (nm) | Emission (nm) |
|---|---|---|
| Fluorecein | 495 | 530 |
| Fluorescein isothiocyanate | 492 | 518 |
| B-Phycoerythrin | 546 | 575 |
| R-Phycoerythrin | 490 | 575 |
| Texas Red | 568 | 590 |
| Rhodamine | 555 | 620 |
| Allophyycocyanin | 650 | 660 |
The antibody is itself conjugated with the fluorochrome and applied directly to a monolayer of cells or to frozen tissue on a slide. When examined with a fluorescence microscope, the antibody labeled with the fluorescent compound identifies the localized antigen.
Unlike direct immunofluorescence, indirect immunofluoresence is a double-layer technique. The unlabeled antibody is applied directly to the tissue substrate and then treated with a fluorochrome-conjugated anti-IgG. There are several advantages to this technique, and it is typically used more frequently than the direct method. Because several fluorescent anti-immunoglobulins can bind to each antibody present in the first layer, this produces brighter fluorescence than in the direct method. It is also more time-efficient since there is only one fluorescent-labeled reagent, the anti-IgG prepared during the lengthy conjugation process. A general protocol for only the indirect immunofluorescence technique is presented here.
Methanol and acetone Fixation result in permeabilized cell preparations. For paraformaldehyde fixed preparations, treat with 0.2% Triton X-100 for 5 min or alternatively with -20°C methanol for 5 minutes.
Note: All incubations should be carried out at RT unless otherwise noted in a humid light-tight box or covered dish to prevent drying and to prevent exposure of fluorochrome to light.
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