New techniques for whole-mount NADPH-diaphorase histochemistry demonstrated in insect ganglia.
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.
Ott
Swidbert R
SR
School of Biological Sciences, Queen Mary, University of London, London, United Kingdom. S.R.Ott@qmul.ac.uk
Elphick
Maurice R
MR
D000818:Animals; D002021:Buffers; D005404:Fixatives; D005557:Formaldehyde; D005629:Frozen Sections; D017952:Ganglia, Invertebrate / Q000033:anatomy & histology / Q000201:enzymology*; D006110:Grasshoppers; D007091:Image Processing, Computer-Assisted; D009252:NADPH Dehydrogenase / Q000378:metabolism*
D002021:Buffers; D005404:Fixatives; D005557:Formaldehyde; D009252:NADPH Dehydrogenase