Immunofluorescence microscopy antibodies

Key Words Macrophage neutrophil phagocytosis immunofluorescence microscopy antibody opsonin. 

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.

Further improvement of microchemical methods for proteinaceous media was based on immunological techniques. The high specificity of the antigen-antibody reaction enables the discrimination of the same protein coming from different species, or the detection of multiple antigens in the same sample. Application to the analysis of artwork has been reported in two types of immunological techniques immunofluorescence microscopy (IFM), and enzyme-linked immunosorbent assays (ELISA) [31]. 

Fig. 4 Immunofluorescence microscopy of Giardia intestinalis showing segregation of mi-tosomes during mitosis. A Interphase, prophase, and C telophase cells. Nuclei were stained by DAPI (blue), mitosomes were detected by an antibody raised against GiiscU (red), and axonemes were visualized by the antibody AXO 49 recognizing polyglycylated carboxy-terminal peptides of a- and fl-tubulin (green). Note the proximity of mitosomes (white arrows) to axonemes...

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. 

A direct detection method was recently developed for these adducts in stratum comeum of human skin based on immunofluorescence microscopy (30). Three partial sequences of keratins containing glutamine or asparagine, adducted with a 2-hydroxyethyl-thioethyl group at the omega-amide function, were synthesized and used as antigens for raising antibodies. After immunization, monoclonal antibodies were obtained with affinity for keratin isolated from human callus exposed to 50 xM sulfur mustard (see Plate 1). In contrast to the immunochemical... 

Proteins can be detected and quantitated by highly specific antibodies monoclonal antibodies are especially useful because they are homogeneous. Enzyme-linked immunosorbent assays and Western blots of SDS-polyacrylamide gels are used extensively. Proteins can also be localized within cells by immunofluorescence microscopy and immunoelectron microscopy. 

Antineutrophil cytoplasmic antibodies (ANCA) are a class of autoantibodies with varied specificities against particular proteins in the cytoplasmic granules of neutrophils and the lysosomes of monocytes. Indirect immunofluorescence microscopy and enzyme immunoassay have defined two types of ANCA patterns one causing cytoplasmic staining (C-ANCA) the other perinuclear staining (P-ANCA) [346]. Greater than 95% of C-ANCA antibiodies are anti-proteinase 3 antibodies (PR3-ANCA) and greater than 95% of P-ANCA antibiodies are anti-myeloperoxidase antibodies (MPO-ANCA) [347]. 

Figure 4. Immunofluorescence microscopy of a cross-section of human skin exposed to saturated sulfur mustard vapor (1 min at 27 °C Ct 1040 mg.min.nr3 A) and of unexposed skin (B), using monoclonal antibody 1H10, directed against sulfur mustard adducts to human keratin, in a 1/50 dilution. The photographs are composed from an image obtained for FITC fluorescence (mainly emanating from the stratum corneum green) and from an image obtained for propidium iodide fluorescence representing DNA (red) in the same cross-section. (Reprinted with permission from G. R van der Schans et al., Chem. Res. Toxicol. 15, 21-25, 2002. Copyright (2002) American Chemical Society).

Fluorescence-based immunolocalization of biomolecules within cells and tissues requires that fluorochromes, or fluorescent markers such as quantum dots, be covalently conjugated to antibodies. Techniques for antibody-fluorochrome conjugation were first devised by A. H. Coons and his associates, who pioneered the use of immunofluorescence microscopy in the 1940s and 1950s. Initially, the conjugation of fluorochrome to antibody was done directly to the antibody with the desired antigenic specificity (1). Subsequently, an indirect method (2) of immunolocalization was introduced that proved advantageous for most routine work. 

---