Tissue substrates staining

Metachromasia, from meta, a change in the kind of, and chroma, color, refers to the qualitative change which occurs in the color of certain dyes when they interact with other substances. The term was originally applied by Paul Ehrlich to the phenomenon of color change when a dye is adsorbed onto a substrate. Thus, cartilege and other mucopolysaccharide-containing tissues are stained red by toluidine blue. Similarly, Hartley observed that when bromophenol blue solution is added to cetrimide solution the color changes from purplish blue to clear blue. 

In addition to the many enzyme systems available, there are with each a series of chromogenic substrate solutions that can be used to create different colors and locations of reaction products. For the peroxidase system, there are numerous oxidizable compounds that precipitate as a permanent color. The most common and still widely used is 3,3 diaminobenzidine tetrahydro-chloride (DAB). This compound precipitates to a golden brown color when in solution with peroxidase and hydrogen peroxide. This brown color has many subtleties and readily stands out in a tissue section. With practice, it is possible to differentiate specific from nonspecific staining patterns just by examining the characteristics of the precipitated pigment. This material is also insoluble in alcohol and xylene, and therefore the tissue may be routinely dehydrated and cleared without loss of chromogen. 

Immunohistochemical stains use antibodies to identify specific constituents in tissue sections. In order to detect the site of reaction, the antibody is labeled with an enzyme that can be reacted with a suitable substrate to give a colored product. The alternative is to use a fluorescent label. The advantage of an enzyme label is that the nuclei can be counter stained thereby revealing the tissue architecture, and that the stain fades slowly, if at all, allowing the slides to be stored. 

In cases where it is necessary to evaluate non-specific binding potentially caused by sources other than the primary antibody, additional patient tissue sections may be stained with selected reagents. For example, tissues may be stained with just the secondary antibody and/or the enzyme followed by application of the substrate/ chromogen. In cases where the suspected non-specific staining may be the result of endogenous enzyme present within the tissue, this can be confirmed by application of the substrate/enzyme only. 

Histochemical techniques for detecting substrate before and after enzyme treatment are extremely useful in studies on cellular structure. One of the oldest histochemical tests utilized saliva to identify suspected glycogen or starch. More definitive results are obtained when thin sections of a tissue are incubated in a buffered solution of purified amylase and stained for poly-utc-glycols. Material stained by periodic acid-Schiff reagent in the control, but not in the section exposed to amylase, is assumed to be glycogen or starch. Two more of the numerous histochemical techniques associated with localization of substrate are—using hya-luronidase to locate hyaluronic acid and chondroitin 4- and 6-sulfates (179) and using neuraminidases to locate sialomucins (180). By use of electron microscopy in combination with the histochemical technique subcellular localization can be obtained. 

Virtually all dentifrice formulations contain abrasive particles, typically composed of amorphous silica, calcium carbonate, alumina or calcium phosphate. The mechanism by which the toothbrush and dentifrice interact to clean the teeth is one of abrasive cleaning. Toothpaste manufacturers thus aim to provide formulations with effective cleaning power, whilst minimising any wear to the underlying substrate. However, in such a system it is inevitable that some degree of abrasivity will be present, as toothpastes without abrasive particles are unable to prevent the build-up of extrinsic stain [12], It is, therefore, important to understand any abrasion a dentifrice product may cause to the hard tissues in the mouth. 

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