Indirect immunofluorescence

Indirect immunofluorescence assay (IFA) 3-6 weeks Plasma Simple to perform, but requires expertise to interpret results... 

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. 

Bronckers, A.J.J., Gay, S., Finkelman, R.D., and Butler, W.T. (1987) Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs Comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement./. Histochem. Cytochem. 35, 825-830. 

Positive enzyme-linked immunosorbent assays are repeated in duplicate and if one or both tests are reactive, a confirmatory test is performed for final diagnosis. Western blot assay is the most commonly used confirmatory test, although an indirect immunofluorescence assay is available. 

As stated above, this approach is applicable only when primary antibodies are raised in different species. For double or multiple indirect immunofluorescence staining with primary antibodies raised in the same species, see Sects. 8.2. and 8.3 below. 

Fig. 9.7 Indirect immunofluorescent staining of SWCNTs-treated F1EK293 cells for day 1-5 by fluorescent microscopy (x 630). Green cadherin red collagen blue cellular nucleus staining with DAPI. The expression levels of cadherin and collagen IV in cells decreased gradually as the culture days increased F1EK293 cells gradually detached from the cell populations as the culture days increased (Grunlan et al., 2004. With permission from Elsevier) (See Color Plates)...

Microtubules in the cytoskeleton and mitotic apparatus are also in a state of dynamic equilibrium and flux with unpolymerized tubulin, and tubulin appears to be an excellent example of the proteins which Pauling (1953) postulated to exist as globular protomers or as insoluble, fibrous, supramolecular structures akin to unpolymerized and polymeric hemoglobin S. The current view of the microtubule cytoskeleton in nondividing celb comes from the development of tubulin-specific antibodies for indirect immunofluorescent localization of microtubules (Fuller et al., 1975 Weber et al., 1975). The general structural features of such cyto-... 

Fig. 2. Microtubule network in fibroblast cells as visualized by use of antibodies directed against tubulin and indirect immunofluorescence techniques. (Courtesy of Dr. William C. Thompson.)...

Apart from antibodies detected by (a) the schizont-infected red cell agglutination test, (b) the agglutination of sporozoites, (c) complement fixation, (d) passive hemagglutination and by the direct and indirect immunofluorescent methods [for review, see reference (V4)], malarial antibodies have also been detected by malarial antigens prepared from heavily infected human placenta, infected human brain, and short-term in vivo cultures of cells from heavily parasitized subjects (Wll) (see Tables 7 and 8). 

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.

The methods outlined below include protocols for direct and indirect immunofluorescence staining, that can be adapted easily for the cell type of interest as indicated in the relevant notes. The principal approaches to flow cytometric analysis, standardization and calibration are then given, followed by two more detailed protocols illustrating quantitation using direct immunofluorescence, and a competitive binding assay, which demonstrates the application of linear amplification of fluorescence. 

Frozen sections, as well as sections of formaldehyde-fixed and paraffin-embedded tissues, can be used for direct or indirect immunofluorescence staining after antigen retrieval using microwave heating. Although frozen sections yield higher sensitivity than that obtained with paraffin sections, the latter approach is necessary for retrospective studies of archival specimens. Moreover, fresh tissue is not always available. 

MICROWAVE HEAT-ASSISTED DOUBLE INDIRECT IMMUNOFLUORESCENCE STAINING... 

Tomehave, D., Hougaard, D. M., and Larsoon, L.-L. 2000. Microwaving for double indirect immunofluorescence with primary antibodies from the same species and for staining of mouse tissues with mouse monoclonal antibodies. Histochem. Cell Biol. 113 19-23. 

---