Alkaline phosphatase localization

Alkaline phosphatase acts on many substrates as well, each precipitating as a different color. For example, a combination of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) results in a permanent blue precipitate at the site of alkaline phosphatase localization. There are other compounds that can also be tried, such as Fast Red TR/Naphthol AS-MX (Sigma, St. Louis, MO), which precipitates as a red color. 

Figure 3 Immuno localization of acetyl esterase. Sections were incubated with antibodies raised against the acetyl esterase, followed by visualization with alkaline phosphatase conjugated secondary antibodies and staining with Fast Red.

Tinglu, G., Ghosh, A., and Ghosh, B.K. (1984) Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold. J. Bacteriol. 159, 668. 

Immunocytochemical methods have been widely applied to visualize proteins, carbohydrates, or lipids in sectioned material. The advantage of using immunocytochemistry is to be able to localize the molecules of interest within the tissue. Several procedures have been described. Basically, these procedures can be split into four main steps that are described in subheadings (1) tissue preparation, (2) the primary antibodies, (3) the visualization of the target, and (4) enhancement of signals with antibody complexes. In addition, a protocol for alkaline phosphatase will be presented in detail in Subheading 5. The terms primary and secondary antibodies refer to the order in which they are applied to the target. The immunocytochemical procedures are not limited to sectioned... 

Plant Cells and Tissues Structure-Function Relationships. Methods for the Cytochemical/Histochemical Localization of Plant Cell/Tissue Chemicals. Methods in Light Microscope Radioautography. Some Fluorescence Microscopical Methods for Use with Algal, Fungal, and Plant Cells. Fluorescence Microscopy of Aniline Blue Stained Pistils. A Short Introduction to Immunocytochemistry and a Protocol for Immunovi-sualization of Proteins with Alkaline Phosphatase. The Fixation of Chemical Forms on Nitrocellulose Membranes. Dark-Field Microscopy and Its Application to Pollen Tube Culture. Computer-Assisted Microphotometry. Isolation and Characterization of... 

Incubate sections for 30 min with (strept)avidin conjugated with a fluorophore or with any enzyme (peroxidase or alkaline phosphatase). For localization of low-density antigens (<10K molecules/cell), VECTASTAIN ABC reagent (http //www.vectorlabs.com/) is preferable. 

Enzymes. Enzymes too are useful labels and several have been employed, with peroxidase and alkaline phosphatase being the most popular to date. One important feature of this technique is for the enzyme to be able to convert a soluble substrate into an insoluble product in order to localize the antigen properly. Several suitable substrates are available with 3 3 -diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) being used with peroxidase and 5-bromo-4-chloro-3-indoylphosphate/nitroblue tetrazolium (BCIP/NBT) with alkaline phosphatase. 

At-MNDP 31-33, 36, 108), whose mechanism of intracellular localization is related to the presence of oncogenically expressed tumor-membrane alkaline phosphatase isoenzymes 42, 108), has been demonstrated strikingly effective in an animal tumor model 33, 34, 38, 39). It has also served as a concomitant analytical probe for identifying the intracellular locus of radiotherapeutic action of this class of drug by a-particle track autoradiography 33,106-109). Phase I and II human therapeutic trials are shortly envisaged 33, 34). 

Described here is an indirect method for detecting two different cellular antigens in acetone-fixed tissue, using a rabbit polyclonal antibody, and a murine monoclonal antibody on the same section. One secondary antispecies antibody is conjugated with alkaline phosphatase, the other with peroxidase, thus resulting in two differently colored products showing the localization of the two antigens (Fig. 1). 

Fig. 1. Double label immunohistochemistry on rai liver. An acetone-fixed rat liver section was incubated with a polyclonal antiserum raised in rabbit to a hepa-tocyte cell surface protein (courtesy of Dr. S. Stamatoglou) and a mouse monoclonal antibody against a bile duct specific cytokeratin (courtesy of Dr. E. B. Lane). The hepatocyte protein was localized by use of a secondary peroxidase-conjugated antibody resulting in a red/brown product (thin arrow). The bile duct cytokeratin was identified by using an alkaline phosphatase-conjugated secondary antibody giving a blue color (thick arrow).

Although the enzyme sediments with intact cells, alkaline phosphatase appears in the supernate when broken cells are centrifuged. Malamy and Horecker (5) discovered that alkaline phosphatase is quantitatively released from the cell when E. coli are converted to spheroplasts by lysozyme and ethylenediaminetetraacetic acid (EDTA) in a sucrose medium. This evidence, supported by the observation that substrates such as glucose 6-phosphate are rapidly hydrolyzed by intact cells with release of most of the phosphate into the medium, led Malamy and Horecker (6) to suggest that alkaline phosphatase is localized in the periplasmic space, a region described by Mitchell (7) as lying between the protoplasmic membrane and the wall layer, and that it is not in association with the wall (8). 

Localization of the enzyme in the periplasmic space is also consistent with the selective release of alkaline phosphatase during growth of an E. coli mutant which is osmotically sensitive because of a defective cell wall (14) and with the fact that phosphate esters which do not penetrate the protoplasmic membrane can be hydrolyzed by intact cells 15). In these latter measurements the activities found with intact cells as compared with equivalent cell extracts varied over wide limits depending upon the substrate and its concentration. This difference was assumed to result from a difference in the ease of penetration of the wall barrier by different phosphate esters. 

At the present time it is not possible to assign a precise function for any alkaline phosphatase. Undoubtedly bone phosphatase is concerned in ossification and two alternative roles have been proposed (1) Precipitation of calcium phosphate is induced by the localized production of high concentrations of Pi owing to phosphatase activity (33) (2) the enzyme permits crystal growth at nucleation sites in the matrix by ensuring the removal and continued absence of PPi which is known to be a crystal poison (60). Other factors must be involved (35) because tissues with high concentrations of alkaline phosphatase (e.g., gut, kidney, and... 

Mouse liver acid phosphatase is localized in the Kupffer cells in contrast to the alkaline phosphatase activity which is largely confined to the endothelial linings of the sinusoids. Under the conditions in which the activity of the reticuloendothelial system is enhanced, both enzymic activities are increased (97, 98). 

An immunohistochemical examination of PSA using polyclonal antibodies by the peroxidase antiperoxidase (PAP) method and by the technique of biotin-streptavidin-alkaline phosphatase has been successfully carried out (Zaviacic et al., 1994). Immunoelectron microscopy in conjunction with the protein A-gold complex can also be used for localizing PSA in human prostate (Sinha et al., 1987). The procedure for immunofluorescence localization of PSA is given below. 

The deposition of limited quantities of hydroxyapatite in extracellular matrix has been observed without bounding cells. Cartilage calcification is such a case where local pH control and Ca2+ are dependent upon diffusion and the rate of mineral deposition is driven by phosphate presentation. Chondrocytes produce alkaline phosphatase that generates the required phosphate, but cartilage is not delimited by any cellular structures and transfer of Ca2+ and H+ is by diffusion from extracellular fluid. 

Immunolocalization is a technique for identifying the presence of a protein within the cell, its relative abundance and its subcellular localization. After suitable preparation of the cells, they are treated with an antibody (the primary antibody) that binds to the protein of interest. An antibody that binds to the primary antibody (the secondary antibody) is then allowed to bind and form an antigen—primary antibody—secondary antibody complex. The detection system generally consists of the formation of a colored insoluble product of an enzymatic reaction, the enzyme, such as alkaline phosphatase or horseradish peroxidase, being covalently linked to the secondary antibody. 

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