Histochemical Analysis of Nitric Oxide Synthase by NADPH Diaphorase Staining

Mirella Gonzalez‐Zulueta1, Valina L. Dawson1, Ted M. Dawson1

1 Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 10.6
DOI:  10.1002/0471140856.tx1006s01
Online Posting Date:  May, 2001
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Abstract

The presence of NOS coincides with that of NADPH diaphorase activity; therefore, NADPH diaphorase staining is widely used to detect NOS‐containing cells in neural and non‐neural tissues. With the appropriate fixation procedure, this method is capable of detecting cells containing any of the NOS isoforms.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cultured cells, free‐floating tissue sections, or slide‐mounted tissue sections
  • Tris‐buffered saline (TBS): 50 mM Tris⋅Cl, pH 7.2 ( appendix 2A)/1.5% (w/v) NaCl
  • 0.2 M sodium phosphate buffer, pH 7.4 (PB; see recipe; prepare fresh)
  • 4% (w/v) paraformaldehyde in 0.1 M PB (see recipe; prepare fresh)
  • 0.2% or 0.4% (v/v) Triton X‐100 in TBS
  • NADPH diaphorase staining solution (see recipe; prepare fresh)
  • TBS containing 0.05% (w/v) sodium azide (optional)
  • Sonicating water bath
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Figures

Videos

Literature Cited

Literature Cited
   Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M., and Snyder, S.H. 1991. Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 7:615‐624.
   Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M., and Snyder, S.H. 1991. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc. Natl. Acad. Sci. U.S.A. 88:7797‐7801.
   Hope, B.T., Michael, G.J., Knigge, K.M., and Vincent, S.R. 1991. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc. Natl. Acad. Sci. U.S.A. 88:2811‐2814.
   Thomas, E. and Pearse, A.G.E. 1994. The solitary active cells. Histochemical demonstration of damage‐resistant nerve cells with a TPN‐diaphorase reaction. Acta Neuropathol. 3:238‐249.
   Vaid, R.R., Yee, B.K., Rawlins, J.N., and Totterdell, S. 1996. NADPH‐diaphorase reactive pyramidal neurons in Ammon's horn and the subiculum of the rat hippocampal formation. Brain Res. 733:31‐40.
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