When processing unfixed tissue for enzyme demonstration, which factor should be avoided?

• A. Heat
• B. Alcohol
• C. Clearing
• D. Infiltration

Answer: (A)

Enzyme activity is heat-labile—exposure to elevated temperatures rapidly denatures proteins and destroys the active site needed for catalysis. When you’re demonstrating enzyme activity in unfixed tissue, preserving those active enzymes is essential, so any processing that involves heat should be avoided. Infiltration steps that use molten paraffin and other hot solvents would denature enzymes and wipe out the very activity you’re trying to observe, making it impossible to visualize enzymatic reactions. Practically, enzyme histochemistry often relies on cold or frozen sections to keep enzymes intact, or at least minimizes thermal exposure during processing.

Alcohol and clearing are harsh solvents and can also affect enzymes, but the most critical, actionable factor to avoid in this context is heat, since even brief heating can obliterate enzymatic activity.