Home › Microbiology › Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues
Microbiology JoVE (Open Access) Citable · DOI

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

DOI: 10.3791/56843-v
What you'll learn
  • ✓ Prepare and apply enzyme cytochemistry reaction buffers to tissue sections
  • ✓ Visualize dehydrogenase activity through microscopic observation and quantification
  • ✓ Determine subcellular localization and kinetics of enzyme activity in tissues
Protocol

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

Difficulty
intermediate
Total time
~2–3 hours per tissue sample set

Steps

1
Prepare enzyme reaction buffer solution

Mix components for the enzyme cytochemistry reaction buffer according to protocol specifications. This buffer is essential for supporting dehydrogenase enzyme activity during incubation.

▶ 00:50
2
Apply incubation medium to tissue sections

Transfer prepared reaction buffer onto tissue sections mounted on microscopy slides. Ensure even coverage of tissue to enable uniform enzyme activity detection.

▶ 03:50
3
Wash microscopy slides to remove excess reagent

Rinse tissue sections with appropriate washing solution to remove unbound reaction components and background signal. This step enhances specificity of enzyme activity visualization.

▶ 05:32
4
Enclose tissue sections for microscopic examination

Mount and seal tissue sections on slides with appropriate coverslip or mounting medium to preserve morphology and prepare for microscopic imaging and quantification.

▶ 06:33
5
Analyze representative results and enzyme localization

Examine microscopical images to identify dehydrogenase activity patterns, determine subcellular localization, and quantify enzyme kinetics in target cells and tissues.

▶ 07:13
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