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Questions and Answers
The optimum pH for the initial substrate enzyme reaction is 7.1.
The optimum pH for the initial substrate enzyme reaction is 7.1.
False
Post incubation coupling has the advantage of avoiding non-specific staining.
Post incubation coupling has the advantage of avoiding non-specific staining.
True
In self-colored substrate technique, the PRP is rendered colorless.
In self-colored substrate technique, the PRP is rendered colorless.
False
The enzyme used as a histochemical reagent must be impure.
The enzyme used as a histochemical reagent must be impure.
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The substrate must be kept localized in the cell by fixation or other means in self-colored substrate technique.
The substrate must be kept localized in the cell by fixation or other means in self-colored substrate technique.
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The concentration of the enzyme and that of the substrate do not affect the histochemical reaction.
The concentration of the enzyme and that of the substrate do not affect the histochemical reaction.
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Post incubation coupling has a number of disadvantages.
Post incubation coupling has a number of disadvantages.
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The optimum conditions of the enzyme action do not need to be ensured for histochemical reactions.
The optimum conditions of the enzyme action do not need to be ensured for histochemical reactions.
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The stability of the enzyme under the conditions of application is not important for histochemical reactions.
The stability of the enzyme under the conditions of application is not important for histochemical reactions.
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Too little of the diazonium salt in the incubating medium will cause inhibition of the rate of hydrolysis of substrate.
Too little of the diazonium salt in the incubating medium will cause inhibition of the rate of hydrolysis of substrate.
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Study Notes
Enzymes in Histochemistry
- Enzymes are proteins that catalyze chemical reactions, essential for metabolic changes in living cells and tissues.
- They are named after their biochemical action with the suffix "(ase)" and have a high degree of specificity, but this degree differs from one enzyme to another.
- Enzymes are sensitive to temperature, pH, and ionic concentration.
Types of Enzymes
- Hydrolases (e.g., phosphatases and esterases)
- Oxidoreductases (e.g., oxidases and dehydrogenases)
- Transferases (e.g., phosphorylases)
- Proteolytic enzymes (e.g., leucine aminopeptidases)
Histochemistry of Enzymes
- Enzymes can be demonstrated histochemically only in fresh tissue.
- They are localized by using their specific substrates, which produce a colored or opaque product.
- Enzymes are highly labile and quickly disappear from tissue, so special precautions must be taken to preserve them in their locality at their optimum activity.
- Cryostat and freeze-drying methods have revolutionized enzyme histochemistry.
- Enzyme histochemistry helps illustrate certain cellular structures, such as:
- Acid phosphatase for lysosomes
- Alkaline phosphatase for membranes
- Dehydrogenase and ATPase for mitochondria
- Esterase for microsomes
Importance of Enzyme Histochemistry
- Enzyme histochemical methods indicate metabolic activities in cells.
- They help compare chemical activities in normal and abnormal cells (e.g., tumors).
- They help identify structures related to certain enzymes when used on ultrathin sections prepared for electron microscopy.
- Enzyme histochemistry is important as a diagnostic tool from a pathological point of view.
Special Precautions for Enzyme Histochemistry
- Tissues must be prepared to preserve enzymes as completely as possible and retain them in their original sites.
- Fixation should not allow diffusion or deterioration of the enzyme.
- Classical embedding techniques are not recommended for most enzymes.
- Control sections are essential to determine specificity and validity of results.
- Ice-cold acetone or alcohol can be used on fresh refrigerated tissues to preserve about 50% of the enzyme.
Enzymatic Reactions
- Enzymatic reactions result in the formation of a Primary Reaction Product (PRP) followed by the production of an opaque or colored Final Reaction Product (FRP).
- Types of enzyme histochemical reactions include:
- Simultaneous capture
- Post-incubation coupling (or post-coupling)
- Self-colored substrate
Types of Techniques Used in Enzyme Histochemistry
- Four basic techniques are available for the demonstration of enzymes:
- Metal precipitation technique
- Simultaneous coupling using diazonium salts
- Post-incubation coupling using diazonium salts
- Self-colored substrate
Conditions for Histochemical Demonstration of Enzymes
- The quantity of the enzyme in a tissue section must be adequate to exceed the threshold of the technique sensitivity.
- The enzyme must not be lost during tissue preparations (fixation, processing, or cutting).
- PRP must be formed without serious diffusion or loss, and FRP must be formed in the accurate site of the original enzyme activity.
- False positive reactions (non-enzymatic) must be eliminated by the use of controls.
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Description
This quiz covers the basics of enzymes, including their role in catalyzing chemical reactions, their high degree of specificity, and their sensitivity to temperature, pH, and ionic concentration.
