Light microscopy immunofluorescence

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. 

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.

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. 

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. 

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. 

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