Antibodies immunofluorescence light

Because of the high specificity of an antibody for its epitope, an antibody raised against a particular protein antigen can be used to determine the location of that antigen in a cell using immunofluorescence light microscopy or immuno-electron microscopy. 

Indirect immunofluorescence assay (IFA) A laboratory test used to detect antibodies in serum or other body fluid. The specific antibodies are labeled with a compound that will make them glow a fluorescent green color when observed microscopically under ultraviolet light. 

Figure 11.2 Morphological differences between human alveolar epithelial cells in primary culture (A and C) and the A549 cell line (B and D). Cells are visualised by light microscopy (A and B) and immunofluorescence microscopy (C and D) using an antibody against a tight junctional protein, occludin.

It should be noted that an alternative approach that avoids some of the disadvantages of immunofluorescence is the use of enzyme-conjugated secondary antibodies (see Chapter 23). Although this approach sacrifices the resolution of a light-emitting source, low-power objectives compatible with thicker whole-mounts give optimal images (13). 

Figure 7-32 Micrograph of a mouse embryo fibroblast was obtained using indirect immunofluorescence techniques.313 The cells were fixed with formaldehyde, dehydrated, and treated with antibodies (formed in a rabbit) to microtubule protein. The cells were then treated with fluorescent goat antibodies to rabbit /-globulins (see Chapter 31) and the photograph was taken by fluorescent light emission. Courtesy of Klaus Weber.

In immunofluorescence microscopy, fluorescent compounds (which absorb light at the exciting wavelength and then emit it at the emission wavelength) are attached to an antibody specific for the subcellular structure under investigation. The antibody is then added to the specimen and allowed to bind. Unbound antibody is removed and the specimen is illuminated at the exciting wavelength, to visualize where the antibody has bound. 

We therefore present two different procedures for fixing and preparing yeast samples for FISH. The first of these is suitable for rDNA hybridization and/or tubulin immunofluorescence (preservation of tubulin requires high concentrations of formaldehyde and rapid fixation). The second protocol is designed for hybridization with sequences of lower copy number and r immunofluorescence with antibodies to nuclear proteins such as RAPl or nuclear pore components. These antigens are extremely sensitive to overfixation, and so the cells must be fixed lightly for immunostaining, then postfixed for hybridization. If possible, use the first procedure because it is simpler and more reliable. 

This protocol was developed for yeast telomere hybridization experiments, in which we wished to localize both telomeric repeats and either RAPl protein or nuclear pore proteins. For such it has been most successful to fix the cells lightly, remove the cell wall, and carry out the immunofluorescence steps first. Following antibody staining, the cells can be postfixed and hybridized without loss of the immunofluorescence signal. The primary fixation method is based on the work done by Davis and colleagues (Davis and Fink, 1990 Loeb et al., 1993) to immunolocalize nuclear pore components in Saccharomyces cerevisiae. 

Antibodies provide a powerful tool to localize antigens in cells or tissues by immunocytochemistiy at the light or electron microscope level. The development of efficient fluorescent dyes which can be coupled to antibodies for their visualization by fluorescence microscopy has pioneered a technology which is known as immunofluorescence microscopy (IFM). IFM is easy to apply to many biological and medical questions, the protocols involved are short, and the development of sophisticated imaging equipment has even made possible the acquisition of quantitative data on the 3D distribution of several antigens in the same specimen. 

Immunofluorescence species-specific secondary antibodies (1 500) conjugated with fluorophores at different excitation wavelengths depending on the microscope (e.g., 488 nm for green or 594 nm for red fluorescence). Fluorochrome-labeled secondary antibodies can be obtained from different suppliers, e.g., Alexa Fluor, Invitrogen or DyLight Fluor, Thermo Fisher Scientific, Waltham, MA (caution light sensitive, store at4°C). 

Wash coverslips in PBS (3x 5 min) and, meanwhile, dilute the secondary antibody 1 40 in the dilution solution. Incubate cover-slips for 2 h in an incubation chamber wrapped with aluminum foil to protect the slides from light. We have employed a Texas Red conjugate to label p75NTR-expressing OECs and Thy 1.1-expressing fibroblasts and a FITC conjugate to label SIOO-expressing OECs. By this means it is possible to use dual color immunofluorescence for the characterization of OECs. 

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