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Histochemical methods for detecting nitric oxide synthase

J E Beesley1

  • 1Wellcome Research Laboratories, Beckenham, Kent, UK.

The Histochemical Journal
|October 1, 1995
PubMed
Summary
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Nitric oxide synthase (NOS) isoforms can be visualized using NADPH-diaphorase (NADPH-d) histochemistry, immunocytochemistry, and in situ hybridization. These methods reveal the enzyme, its associated protein, and mRNA, offering valuable insights into NOS biology.

Area of Science:

  • Neuroscience
  • Cell Biology
  • Biochemistry

Background:

  • Nitric oxide synthase (NOS) exists in three main isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS).
  • Accurate localization of NOS is crucial for understanding its diverse physiological roles.
  • Existing techniques for visualizing NOS have limitations in specificity and resolution.

Purpose of the Study:

  • To evaluate and compare the utility of NADPH-diaphorase (NADPH-d) histochemistry, immunocytochemistry, and in situ hybridization for localizing NOS.
  • To determine the ultrastructural and cellular distribution of NOS isoforms in various tissues.
  • To highlight the complementary roles of these techniques in studying NOS biology.

Main Methods:

  • NADPH-diaphorase histochemistry to visualize the enzyme's reaction product.

Related Experiment Videos

  • Immunocytochemistry with nickel enhancement to detect NOS protein.
  • In situ hybridization using radio-labelled probes to identify NOS mRNA.

    • Main Results:

    • NADPH-d histochemistry showed a blue deposit, with ultrastructural localization in rat hippocampus predominantly on endoplasmic reticulum membranes.
    • Immunohistochemistry revealed nNOS in rat brain dendrites and soma, and patchy distribution in guinea-pig ileum myenteric neurons without specific organelle association.
    • In situ hybridization detected nNOS mRNA as dense granules throughout neurons in rat dorsal root ganglia.

    Conclusions:

    • NADPH-d histochemistry, immunocytochemistry, and in situ hybridization are valuable complementary techniques for NOS localization.
    • NADPH-d detects an enzyme associated with NOS, immunocytochemistry detects the NOS molecule itself, and in situ hybridization detects its mRNA.
    • Carefully controlled application of these methods provides comprehensive insights into NOS distribution and biology.