of reaction. In a typical reaction to display a hydrolytic enzyme, the tissue section is placed in a solution containing a substrate (AB) and a trapping agent (T) that precipitates one of the products as follows: where AT is the trapped end product and B is the hydrolyzed substrate.
By using such methods, the lysosome, first identified in differential centrifugation studies of cells, was equated with a vacuolar component seen in electron micrographs. In lightly fixed tissues, the acid hydrolases and esterases contained in lysosomes react with an appropriate substrate. The reaction mixture also contains lead ions to precipitate (e.g., lead phosphate derived from the action of acid phosphatase). The precipitated reaction product can then be observed with both light and electron microscopy.
Similar light and electron microscopy histochemical procedures have been developed to demonstrate alkaline phosphatase, adenosine triphosphatases (ATPases) of many varieties (including the Na /K -ATPase that is the enzymatic basis of the sodium pump in cells and tissues), various esterases, and many respiratory enzymes (Fig. 1.3).
The specificity of a reaction between an antigen and an antibody is the underlying basis of immunocytochemistry.
Antibodies, also known as immunoglobulins, are glyco proteins that are produced by specific cells of the immune system in response to a foreign protein, or antigen. In the laboratory, antibodies can be purified from the blood and conjugated (attached) to a fluorescent dye. In general, ﬂuorescent dyes (ﬂuorochromes) are chemicals that absorb oped to study DNA increases in developing cells and to analyze ploidy–that is, the number of times the normal DNA content of a cell is multiplied (a normal, nondividing cell is said to be diploid; a sperm or egg cell is haploid). Two techniques, static cytometry for tissue sections and ﬂow cytometry for isolated cells, are used to quantify the amount of nuclear DNA. The technique of static cytometry of Feulgen-stained sections of tumors uses microspectrophotometry coupled with a digitizing imaging system to measure the absorption of light emitted by cells and cell clusters at 560-nm wavelength. In contrast, the flow cytometry technique uses instrumentation able to scan only single cells flowing past a sensor in a liquid medium. This technique provides rapid, quantitative analysis of a single cell based on the measurement of fluorescent light emis-
Schiff reagent to give the distinctive magenta color. The reaction of the Schiff reagent with DNA is stoichiometric, meaning that the product of this reaction is measurable and proportional to the amount of DNA. It can be used, therefore, in spectrophotometric methods to quantify the amount of DNA in the nucleus of a cell. RNA does not stain with the Schiff reagent because it lacks deoxyribose.
Enzyme digestion of a section adjacent to one stained for a specific component—such as glycogen, DNA, or RNA— can be used to confirm the identity of the stained material.
Intracellular material that stains with the PAS reaction may be identified as glycogen by pretreatment of sections with diastase or amylase. Abolition of the staining after these treatments positively identifies the stained material as glycogen.
Similarly, pretreatment of tissue sections with deoxyribonuclease (DNAse) will abolish the Feulgen staining in those sections, and treatment of sections of protein secretory epithelia with ribonuclease (RNAse) will abolish the staining of the ergastoplasm with basic dyes.
Histochemical methods are also used to identify and localize enzymes in cells and tissues.