TECHNICAL NOTE |
Correspondence to: Nico P. Dantuma, Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden..
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Summary |
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We present a modified diaminobenzidine (DAB) photoconversion method that enables staining of internalized DiI-labeled lipoproteins without the apparent punctate background staining that was observed with the original DAB photoconversion method. This is illustrated by the localization of DiI-labeled insect lipoproteins in natural recipient cells that internalize these lipoproteins by receptor-mediated endocytosis. Exposure to DiI-excitation light of cells that had been incubated with DiI-labeled lipoproteins yielded a light- and electron-dense DAB reaction product. In addition to the expected staining, an apparent punctate background staining of vesicular structures hindered proper identification of DiI-containing vesicles because these background-stained vesicles were indistinguishable from putative late endosomal and lysosomal structures at the electron microscopic level. This background staining was completely abrogated by inhibition of peroxisomal catalase with aminotriazole. The conversion of DAB by the emitted light of DiI was not affected by aminotriazole. We conclude that specific staining of DiI-labeled intracellular structures can be achieved with the modified DAB photoconversion method reported here. (J Histochem Cytochem 46:10851089, 1998)
Key Words: photoconversion, diaminobenzidine, aminotriazole, DiI, electron microscopy, endocytosis, peroxisome, lipophorin, insect
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Introduction |
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We attempted to apply DAB photoconversion for visualization of DiI-labeled insect lipoproteins internalized by the natural recipient tissue, the fat body. In preliminary studies we noted that the original DAB photoconversion technique cannot be used for this purpose, because of a prominent punctate background staining that seriously hindered identification of DiI-containing structures. Similar staining and its implications have been reported previously for DAB photoconversion by DiI-labeled mammalian very low-density lipoproteins and low-density lipoproteins in macrophages (
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Materials and Methods |
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Animals
Migratory locusts, Locusta migratoria, were reared under crowded conditions as described previously (
Isolation and Labeling of Lipoproteins
HDLp was isolated from hemolymph samples by density gradient ultracentrifugation (
DAB Photoconversion
Freshly dissected fat body tissue was rinsed in Buffer A (10 mM HEPES, 150 mM NaCl, 10 mM KCl, 4 mM CaCl2, 2 mM MgCl2, pH 7.0). The tissue was incubated in Buffer A + 0.3 mg/ml DiI-labeled HDLp for 90 min at 30C. Subsequently, the fat body tissue samples were rinsed three times for 10 min in Buffer B (0.1 M NaH2PO4/Na2HPO4, pH 7.4) at 0C, fixed for 3 days in 2% paraformaldehyde and 0.5% glutaraldehyde in Buffer B, and rinsed again three times for 10 min in Buffer B, both at 4C. The tissue samples were embedded in 7.5% agar, from which 50-µm vibratome sections were cut. The sections were incubated overnight in 2% paraformaldehyde in Buffer B and rinsed three times for 10 min in Buffer B, both at 4C. Subsequently, the vibratome sections were preincubated in Buffer C [Buffer B + 20 mM 3-amino-1,2,4-triazole (ATA; Sigma, St Louis, MO) + 0.001% H2O2] for 1 hr at room temperature. In a subsequent preincubation, the sections were incubated for 1.5 hr at 0C in Buffer C containing 1.5 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) filtered over a 0.22-µm filter. Finally, the sections were photoconverted for 1 hr in the same prechilled (0C) buffer using a conventional fluorescence microscope (Axioskop; Zeiss, Oberkochen, Germany) with a rhodamine filter setting (Zeiss; BP 520-560, FT 580, LP 590), a 50-W HBO light source, and a x10 objective. Every 15 min during this incubation, fresh prechilled DAB-containing Buffer C was added. The illuminated area of the vibratome tissue section, which could be recognized by the dark DAB product, was excised. These excised tissue samples were rinsed three times for 10 min in Buffer B at 0C, postfixed for 30 min in Buffer B + 1% OsO4 at 4C, and rinsed again for three times for 10 min in distilled water. After sequential dehydration in a graded ethanol series and propylene oxide, the tissue was embedded in an epoxy resin (glycide ether 100; Merck, Darmstadt, Germany). Semi- and ultrathin sections were cut and examined by light microscopy (Axioskop; Zeiss) and transmission electron microscopy (EM10A; Zeiss), respectively.
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Results |
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We attempted to visualize DiI-labeled HDLp in fat body tissue of young adult locusts 4 days after imaginal ecdysis, a developmental stage for which we have previously shown that the fat body cells internalize HDLp (
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In the samples that had been incubated with DiI-labeled HDLp, in addition to the background-stained vesicles typical endosomal structures were found to contain DAB reaction product, as discussed below. Because the background-stained vesicles could not be distinguished from putative late endosomal or lysosomal structures containing DiI-labeled HDLp, it was a prerequisite to reduce the background staining.
No punctate autofluorescence was observed in non-photoconverted control fat body samples that had not been incubated with DiI-labeled HDLp or had been incubated with DiI-labeled HDLp in the presence of an excess of unlabeled HDLp when examined with fluorescence microscopy (
In contrast to the complete abrogation of the punctate background staining by ATA, this catalase inhibitor did not affect the actual DAB photoconversion induced by the DiI-labeled HDLp. Fat body tissue that had been incubated with DiI-labeled HDLp still revealed an apparent punctate staining in the presence of ATA (Figure 1C).
Transmission electron microscopy of photoconverted fat body samples incubated with DiI-labeled HDLp in the presence of ATA revealed that the punctate staining represents labeling of intracellular vesicles, which is in good agreement with receptor-mediated uptake of HDLp (Figure 3A). Occasionally, in some of these vesicles spherical structures with dimensions identical to those of lipoprotein particles could be distinguished. These putative lipoproteins were visible either as dark spherical particles (Figure 3B) or as typically negative-stained particles (Figure 3C), similar to those observed after negative staining of HDLp (
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Apparently, the emitted light of DiI converted DAB only in a small area surrounding the fluorescent dye. Moreover, this area did not exceed the lumen of the labeled vesicles. Even when vesicles were heavily stained, no labeling was observed outside the lumen.
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Discussion |
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Visualization of a fluorescent signal by photoconversion of DAB into an electron-dense product by the emitted light of fluorescent dyes has been used mainly in neuroanatomical studies, in which different dyes, such as Lucifer Yellow (
To our knowledge, only a single study has been published (
The background staining of peroxisomes on DAB photoconversion appears not to be as general as might be expected, because in a previous study (
ATA did not affect the photoconversion of DAB because the distribution of the electron-dense DAB product, present on photoconversion by internalized DiI-labeled HDLp, corresponded with the previously reported staining pattern observed with fluorescence microscopy (
Photoconversion was established only in close proximity of the labeled lipoproteins according to the occasionally observed individual staining of lipoproteins and the fact that staining of vesicles was always restricted to the lumen and never diffused into the cytosol. This demonstrated that the DAB reaction product marked the precise location of the internalized fluorescently labeled lipoproteins.
In conclusion, we have demonstrated that DAB photoconversion can be used to localize a fluorescent ligand in the endosomal compartment at the electron microscopic level. Addition of ATA during DAB photoconversion is required for appropriate identification of labeled organelles in tissues that express high levels of catalase. Because ATA did not affect the true DAB photoconversion, it may be advisable to include this agent routinely when visualization of an intracellular dye is pursued.
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Acknowledgments |
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Supported by the Life Science Foundation (SLW), which is subsidized by the Netherlands Organization for Scientific Research (NWO) (SLW-805.27.062).
We thank Drs Henk van den Bosch and Fred Wouters for helpful suggestions on the inhibition of peroxisomal staining.
Received for publication November 10, 1997; accepted April 15, 1998.
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Literature Cited |
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