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. 

Fluorescent dyes. Immunofluorescence using fluorescein or rhodamine has been very successfully employed for immunohistochemistry. Fluorescein, when illuminated with UV light, emits a characteristic green fluorescence while rhodamine gives an orange colour. 

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.

Figure 25. A-D Immunofluorescence staining of vmculrn in vascular smooth muscle cells on day 3 after seeding on polymeric surfaces (medium supplemented with 10% fetal bovine serum). A poly(DL-lactic acid), PDLLA B block copolymer of poly(DL-lactic acid) and poly (ethylene oxide) (PEO), PDLLA-6-PEO C, E PDLLA-6-PEO with 5% GRGDSG-PEO-6-PDLLA D, F PDLLA-6-PEO with 20% GRGDSG-PEO-6-PDLLA. E, F Immunoperoxidase staining of bromodeoxyuridine (arrows) incorporated into DNA newly synthesized in vascular smooth muscle cells cultured for 3 days in serum-free medium on PDLLA-Z)-PEO with 5% (E) or 20% (F) GRGDSG-PEO-6-PDLLA. Cells counterstained with light green. Bar=100 pm [41].

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.

Minimal change disease is the most common cause of nephrotic syndrome in children, presenting typically with rapid onset of mostly steroid-sensitive nephrotic syndrome, usually with selective proteinuria (albuminuria). Light-microscopic morphology of the kidney is normal and immunofluorescence is negative. Foot process effacement on electron microscopy is the only observed pathology. 

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. 

This variation of immunofluorescence microscopy uses a laser to focus light of the exciting wavelength on to the specimen so that only a thin section of it is illuminated. The laser beam is moved through the sample, producing a series of images which are then reassembled by a computer to produce a three-dimensional picture of the specimen. 

Male Wistar-Kyoto rats weighing 150 g receive either continuous administration of the test drug by an osmotic pump (ALZA Co., Palo Alto, USA) or saline. Twenty-four hours later, the rats are injected with 1 ml of nephrotoxic serum. At 9, 12, and 14 days, urine samples are collected and urinary protein levels are measured using the Lowry method. At 14 days the rats are sacrificed under ether anesthesia, and both kidneys are removed. Portions of these tissues are processed for light microscopy, immunofluorescence staining and immunoperoxidase staining. 

Rocamora A, Matarredona J, Sendagorta E, Ledo A. Sweat gland necrosis in drug-induced coma a light and direct immunofluorescence study. J Dermatol 1986 13(l) 49-53. 

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