ARTICLE |
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
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Summary |
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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 51:523532, 2003)
Key Words: neuroanatomy, nitric oxide synthase, fixation, tissue clearing, locust, central nervous system, method, 3D imaging
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Introduction |
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NADPH-diaphorase (NADPHd) activity in formaldehyde-fixed tissue can be largely attributed to nitric oxide synthase (NOS), the enzyme responsible for biosynthesis of the diffusible free radical messenger molecule nitric oxide (NO;
Even when NOS antibodies yield well-defined staining, NADPHd is a valuable tool for independent evaluation. For example, affinity-purified uNOS antibodies give positive staining in locust skeletal muscle but no corresponding NADPHd staining has been found, suggesting that in locust muscle the uNOS antibodies may crossreact with material other than NOS (
Although high anatomic detail usually requires the NADPHd staining to be performed on frozen sections, it has also been applied to unsectioned specimens (whole-mounts; e.g., Xenopus tadpole central nervous system,
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Materials and Methods |
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Adult locusts (Schistocerca gregaria, gregarious phase) were bought from Blades Biological (Cowden, Kent, UK). Reagents were obtained from either SigmaAldrich, Poole, UK or BDH Chemicals, Poole, UK, unless stated otherwise. Thoracic nerve cords were dissected out in HEPES-buffered saline (
Fixation
Two different fixatives were compared, buffered formaldehyde (BF) and methanol/formalin (MF).
BF Fixation. Buffered formaldehyde (BF; 4% formaldehyde in 0.1 M phosphate buffer, pH 7.4) was prepared from paraformaldehyde powder, aliquotted, and stored at 20C; aliquots were thawed immediately before use. Ganglia were fixed in BF for 2 hr at 4C and then washed twice for 15 min in PB at 4C. This was followed by overnight permeabilization on ice in 0.2% Triton X-100 in 0.1 M Tris buffer, pH 8.0 (Tris-Tx).
MF Fixation.
Methanol/formalin (MF;
Whole-mount NADPHd Staining
Three alternative NADPHd staining regimens were used: direct staining, staining after cold preincubation, and staining after acidic preincubation.
Direct Staining. Ganglia were directly incubated in an NADPHd staining solution of 0.2 mM ß-NADPH and 0.2 mM nitroblue tetrazolium (NBT) in Tris-Tx for 13 hr at RT.
Staining After Cold Preincubation. Ganglia were incubated in the same NADPHd staining solution as for direct staining, but on ice rather than at RT, for 3 hr under constant agitation. Only very weak staining developed during this preincubation due to low enzyme activity at 0C. Staining was then triggered by bringing the preparations to RT and was allowed to develop for 3060 min.
Staining After Acidic Preincubation. This is effectively a modified cold preincubation that is carried out at low pH to further reduce enzyme activity. Overnight incubation in acetate buffer, pH 5.0, was omitted. Instead, ganglia were preincubated overnight on ice with 0.2 mM NADPH and 0.2 mM NBT in 0.1 M Sørensen phosphate buffer, pH 5.0, containing 0.2% Triton X-100 (phosphate buffer was used because NADPH appeared unstable in acetate buffer). Development was then triggered by placing the tissue in the pH 8.0 staining solution (see above; 0.1 M Tris buffer) at RT.
Clearing of Whole-mounts
The staining reaction was stopped by extensive washes in distilled water. Whole-mounts were then cleared using one of the following three approaches: glycerol clearing, ethanol/xylene clearing, and methanol/cedar oil clearing.
Glycerol Clearing.
Ganglia were cleared and subsequently mounted on slides, in a 9:1 mixture of glycerol and 0.1 M phosphate buffer, pH 7.4 (e.g.,
Ethanol/Xylene Clearing.
Ganglia were mounted on slides in DPX after dehydration in ethanol and clearing in xylene or methyl salicylate (e.g.,
Methanol/Cedar Oil Clearing. We developed this new technique to avoid the shortcomings in the two above approaches. Ganglia were transferred directly into a freshly prepared 3:1 mixture of absolute methanol and glacial acetic acid for 1530 min, followed by three times for 1530 min in absolute methanol and 3060 min in cedar oil (Cedarwood Oil for Clearing; RA Lamb, London, UK). Preparations were then mounted permanently in cedar oil on cavity slides.
Frozen Sections
Some ganglia that had been whole-mount-stained for NADPHd were subsequently sectioned rather than cleared. These ganglia were cryoprotected at RT in 20% sucrose in 0.1 M phosphate buffer containing 0.005% sodium azide, embedded in Jung Tissue Freezing Medium (Leica; Nussloch, Germany), and frozen on the surface of liquid nitrogen. A Leica CM1800 cryostat was used to cut 30-µm frozen sections, which were collected on chromalum/gelatin-coated slides and mounted in glycerol jelly.
Imaging
Whole-mounts and sections were viewed under a Leica DMRA2 compound microscope fitted with a motorized Z-stage. Digital images were captured using a Retiga 1300 12-bit monochrome camera (QImaging; Burnaby, BC, Canada) and QCapture 1.1.6 (QImaging) or OpenLab 3.0.8 (Improvision) software running on a Macintosh G4 computer (Apple Computer; Cupertino, CA). Color images were acquired using the camera in combination with an RGB Liquid Crystal Color Filter module (QImaging). All further processing was done in ImageJ 1.27z (Wayne Rasband, National Institutes of Health, <http://rsb.info.nih.gov/ij/; ImageJ is in the public domain) and Photoshop 6.0 (Adobe Systems; San Jose, CA) software. For Fig 1A and Fig 1D, the captured 12-bit images were log-transformed before mapping their dynamic range to 8 bit; multiple focal planes were combined in Photoshop using the "Darken" layer mode. For Fig 1B and Fig 1C, Fig 1E and Fig 1F, the dynamic range of the captured 12-bit images was mapped linearly to 8 bit.
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The 3D image (anaglyph) in Fig 2G was constructed from 75 focal planes taken at 5-µm intervals along the Z-axis (dorsoventral axis of the ganglion). The XY-resolution was increased by using a x20 objective and capturing six Z-stacks that overlapped in the XY-plane and together covered the entire ganglion. The 12-bit camera frames were log-transformed and downsampled to 8-bit resolution, and each set of six frames belonging to one focal plane was merged into a single high-resolution image. Next, each focal plane was brightness-inverted, and out-of-focus blur was reduced by Rolling Ball background subtraction (75 pixel radius) in ImageJ. Labels were applied to individual focal planes. The anaglyph was then constructed from two Brightest Point projections that simulate a binocular ventral view with an interocular viewing angle of 12°.
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Results and Discussion |
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NADPHd histochemistry in whole-mounts is severely limited by the poor penetration of the staining reagents NADPH and NBT, by NOS-unrelated NADPHd, and by tissue opacity. Naturally, these limitations are the more pronounced the thicker the specimens are. Background, for example, adds up over the thickness of the preparation and is therefore much more critical in whole-mounts than in sections. The problems are readily demonstrated in the metathoracic ganglion of the locust Schistocerca gregaria. In adult locusts, this ganglion measures about 1800 x 1400 x 800 µm (unfixed). In addition to its considerable size, two previously published studies make this preparation a very instructive test object. The first report on NADPHd in locust thoracic ganglia was based on whole-mount preparations that showed stained neuronal cell bodies (
Whole-mount NADPHd After BF Fixation
Fig 1A shows a typical example of the whole-mount staining in the metathoracic ganglion of an adult locust after application of "conventional" techniques (BF fixation, direct staining, glycerol clearing; for details see Materials and Methods). This experiment closely replicates the results of
Like
Whole-mount NADPHd After MF Fixation
Poor penetration and background are thus mutually aggravating because the latter is amplified by the long incubations necessitated by the former. This prompted us to explore whether fixation in methanol/formalin (MF) might improve the results in whole-mounts. We have recently developed MF fixation as a means to preserve the strongly formaldehyde-sensitive NOS/NADPHd of the cockroach Periplaneta americana (
MF fixation yielded pronounced improvements in the detection of NADPHd in intact thoracic ganglia. First, the staining extended much further into the neuropil, reflecting the facilitated penetration of the staining reagents in MF-fixed tissue. However, direct staining (as defined in Materials and Methods) still failed to reveal the NADPHd-positive fibers in the core of the ganglion with incubation times that avoid overstaining the periphery (not shown). Only by combining MF fixation with cold or acidic preincubation was it possible to reveal the NADPHd fiber architecture evenly throughout the ganglion (Fig 1D and Fig 2D2G; the fainter appearance of the staining "inside" the image in Fig 2G is largely an artifact of the Rolling Ball background subtraction used to reduce out-of-focus blur). The rationale behind cold preincubation is that diffusion is linearly dependent on temperature, whereas enzyme reactions have higher-order thermal kinetics. Low temperatures thus favor diffusion over enzyme activity and allow incubation in the staining reagents without concomitant development of staining. Slight staining that may develop during prolonged cold preincubation can be completely suppressed by acidic preincubation, which also exploits the pH dependency of NOS enzyme activity (cf.
Second, and equally important, the signal-to-background ratio in intact ganglia was far better after MF than after BF fixation. NADPHd-positive cell bodies and a plethora of sharply stained arborizations were revealed within a virtually unstained matrix (compare Fig 2D2F to Fig 2A2C). As expected from previous analysis in sections (
Clearing the Tissue for Whole-mount Viewing
NADPHd staining that extends into the depth of the tissue is of little use if the tissue cannot be cleared sufficiently for whole-mount viewing. Buffered glycerol induced heavy anisometric shrinkage and the ganglia collapsed dorsoventrally (this flattening is not very apparent from the ventral view in Fig 1A, but note the resultant folding of the surface in Fig 2B). This finding is in agreement with
We therefore developed a new clearing technique (methanol/cedar oil clearing; Fig 1D and Fig 2D2G) on the basis of three observations. First, the blue form of NBF, which accounts for the selective staining, is very stable in methanol, while pink background NBF is extracted by it. Second, the swelling action of acetic acid counteracts the morphological distortions that occur during dehydration. Third, cedar oil makes an excellent clearing medium which, unlike xylene and methyl salicylate, does not dissolve the NBF. For dehydration, the ganglia were transferred from water directly into a single bath of methanol/acetic acid 3:1 for 1530 min, followed again directly by three baths of 1530 min in absolute methanol. This rapid and convenient procedure gave better preservation of morphology than dehydration through a finely graded methanol series (10% steps, 30 min each). After clearing in cedar oil, the preparations showed 3040% isometric shrinkage (compared to unfixed ganglia in physiological saline) but virtually no deformation artifacts.
The combination of MF fixation, cold or acidic preincubation, and methanol/cedar oil clearing results in NADPHd whole-mount staining of unprecedented anatomic resolution. Particularly under differential interference contrast illumination, the preparations yield images of spectacular definition that could be mistaken for physical sections (Fig 2D2F), even when focusing deep into the ganglion (Fig 2F). However, the greatest advantage lies in the rich 3D detail, which is only poorly conveyed by 2D images. The 3D anaglyph in Fig 2G has been created from 75 focal planes taken through an intact metathoracic ganglion. Although it must necessarily fall short of direct inspection through the microscope, it demonstrates the power of the techniques presented here. Moreover, it both complements and condenses into a single three-dimensional image the neuroanatomical analysis of
The improvements in whole-mount NADPHd staining shown here are in part due to the use of MF in place of BF as a fixative, which improves whole-mount staining for two independent sets of reasons. First, MF fixation enhances the histochemical detection of putatively NOS-related NADPHd per se. Second, it renders the tissue much more permeable to the staining reagents. In a previous report (
The improved penetration of the staining reagents after MF fixation and cold preincubation, and the advantages of methanol/cedar oil clearing, do not depend on species- or tissue-specific enzyme properties and are hence universally applicable in vertebrate and invertebrate tissue. We have applied the method successfully to other parts of the locust nervous system, including abdominal ganglia and the brain, and to the nervous system of other invertebrates, including crayfish (
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Acknowledgments |
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Supported by grant S11816 from the BBSRC to MRE.
Received for publication August 5, 2002; accepted October 25, 2002.
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