Enzyme Stains

If you are planning on taking the HTL exam, you will need to be familiar with enzyme histochemistry. Enzyme histochemistry is used to visualize the activity of an enzyme through the use of chemical reactions. So, first of all, what is an enzyme? Enzymes are molecules produced by the body to help speed up reactions. Enzyme reactions are sometimes helped along by cofactors, typically either a metal, such as zinc, magnesium, or potassium, or a coenzyme such as biotin. Cofactors make the reaction more stable and consistent.

It is necessary to learn the chemistry of enzymes for histology, as the wrong conditions can render an enzyme ineffective and unable to be visualized. For example, many are inactivated by heat. Exposure to heat during paraffin processing can denature the enzyme so it does not fit its substrate, which is why these specimens are usually frozen sectioned. Enzymes are also typically sensitive to pH changes, with most working best at a neutral pH.

So, what types of enzyme stains are there and what are they used for?

Myofibrillar ATPase is used for skeletal muscle fiber typing. There are two types of muscle fiber, type I (slow twitch) and type II (fast twitch). As you may have guessed if you have done any athletic training, slow twitch muscle fibers are more efficient at generating long term ATP and can go for a long time without fatigue, whereas fast twitch muscle fibers fire more rapidly and generate short bursts of energy. In this reaction ATP is the substrate, and myosin is the enzyme. The end product, after activation by calcium and the subsequent Gomori Metal Precipitate reaction, is cobalt sulfide, an insoluble black precipitate. Because the type II muscle fibers use up ATM faster, they create more precipitate making the type II fibers appear darker in the stain result.

Cytochrome Oxidase: Cytochrome oxidase is an enzyme in the inner membrane of the mitochondria that catalyzes the reaction of the electrons remaining towards the end of the mitochondrial electron transport chain, with oxygen that is essential in creating energy. Cytochrome oxidase is often used in histochemistry to evaluate brain function, particularly in the study of Alzheimer’s disease. It is also used to look at problems with mitochondria in other organs such as kidneys, heart, and liver, which can be the result of genetic diseases or a side effect of some drugs such as those used for HIV or cancer.

Succinic Dehydrogenase is an enzyme generally used to locate mitochondria. During this reaction, Succinic Dehydrogenase oxidizes succinate and produces fumarate. Nitroblue tetrazolium is used to visualize the reaction, which is supposed to demonstrate how many mitochondria there are and their activity levels.

Succinic Dehydrogenase- Beaumont Health

Esterase: Esterase is an enzyme that contributes to the hydrolysis of ester. It is generally used in enzyme histochemistry to stain denervated muscle fibers, motor end plates (where motor neuron meets skeletal muscle fiber), lysosomes, and macrophages which will appear red/brown while normal muscle is pale yellow. The substrate here is alpha-naphthyl acetate.

Acid Phosphatase is an enzyme found in lysosomes. In histochemistry, sites of acid phosphatase activity are stained red when incubated in a solution of azotized pararosaniline and napthol AS‐Bl phosphoric acid. As the name acid implies, this works best at a pH between 4.5-5.9.

To learn more about enzyme histology, and other topics covered on the HTL exam, check out NSH’s HTL Prep Course module on elearn.nsh.org.








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The National Society for Histotechnology is a professional member organization for individuals actively involved in the histology profession. NSH has over 3,000 members worldwide, and is the leading provider of histology focused continuing education.  



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