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Journal of Histochemistry and Cytochemistry, Vol. 51, 523-532, April 2003, Copyright © 2003, The Histochemical Society, Inc.

ARTICLE

New Techniques for Whole-mount NADPH-diaphorase Histochemistry Demonstrated in Insect Ganglia

Swidbert R. Otta and Maurice R. Elphicka
a School of Biological Sciences, Queen Mary, University of London, London, United Kingdom

Correspondence to: Swidbert R. Ott, School of Biological Sciences, Queen Mary, University of London, Mile End Road, London  E1 4NS, UK. E-mail: S.R.Ott@qmul.ac.uk

Fixation-resistant NADPH-diaphorase (NADPHd) activity is used widely as a marker for nitric oxide synthase (NOS). In frozen sections, NADPHd histochemistry yields high anatomic definition. In whole-mounts, however, poor penetration of the reagents, background staining, and tissue opacity severely limit its application. Here we report a combination of new methods that significantly improves whole-mount NADPHd staining. We demonstrate these methods in the thoracic ganglia of a large insect, the locust Schistocerca gregaria, in which NADPHd has been analyzed previously using both whole-mounts and serial section reconstructions. The penetration of the staining reagents was markedly improved after fixation in methanol/formalin compared to phosphate-buffered formaldehyde. Methanol/formalin also reduced nonspecific NADPHd and enhanced the selective staining. Penetration was further enhanced by incubation regimens that exploit the temperature- or pH-dependence of NADPHd. In combination with methanol/formalin fixation, this permitted staining to develop evenly throughout these comparatively large invertebrate ganglia. These improvements were complemented by a new clearing technique that preserves the NADPHd staining, gives excellent transparency, and avoids distortion of specimen morphology. The new methods revealed the three-dimensional architecture of NADPHd expression in locust ganglia in unprecedented detail and may similarly improve whole-mount detection of NADPHd in other invertebrate and vertebrate preparations.

(J Histochem Cytochem 51:523–532, 2003)

Key Words: neuroanatomy, nitric oxide synthase, fixation, tissue clearing, locust, central nervous system, method, 3D imaging


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