ARTICLE |
Correspondence to: A. Nanci, Université de Montréal, Faculty of Dentistry, Dept. of Stomatology, PO Box 6128, Station Centre-Ville, Montreal, QC, Canada H3C 3J7. E-mail: Antonio.Nanci@UMontreal.CA
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Summary |
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Methylmethacrylate (MMA) embedding of undecalcified bone is routinely employed for histomorphometric analyses. Although MMA-embedded bone has been used for immunolabeling at the light microscopic level after removal of the resin, there are no such reports for electron microscopy. The aim of the present study was to determine whether MMA embedding can be used for ultrastructural immunolabeling and how it compares to LR White (LRW), an acrylic resin frequently used for immunocytochemistry of bone. Rat tibiae were fixed by vascular perfusion with aldehyde and embedded either in MMA or LRW resin. Thin sections were processed for postembedding protein Agold immunolabeling with antibodies to rat bone sialoprotein (BSP) and osteopontin (OPN). The density of gold particles over bone was quantified. The density and distribution of immunolabeling for BSP and OPN respectively, were comparable between MMA and LRW. These results indicate that MMA performs as well as LRW for the ultrastructural immunolabeling of noncollagenous bone matrix proteins. (J Histochem Cytochem 51:6167, 2003)
Key Words: bone, bone sialoprotein, immunocytochemistry, LR White resin, methylmethacrylate osteopontin
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Introduction |
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ACRYLIC RESINS are widely used for immunolabeling because they permit a good balance between tissue preservation and retention of antigenicity. Among them, methylmethacrylate (MMA) offers a number of advantages. Tissue penetration is good, a particularly important property for larger specimens and calcified tissue samples. MMA can be removed easily and completely from tissue sections, resulting in superior staining characteristics and good morphological detail (
The glycolated form of methacrylate (glycolmethacrylate, GMA) is employed for both light and electron microscopic immunolabeling. GMA is a bifunctional methacrylate that forms crosslinks during polymerization. Consequently, it cannot be dissolved out of sections (
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Materials and Methods |
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Tissue Processing
Male Wistar rats weighing 100 ± 10 g (Charles River Canada; St-Constant, QC, Canada) were anesthetized with chloral hydrate (0.4 mg/g body weight). For embedding in LRW (London Resin Company; Berkshire, UK), rats were perfused for 20 min with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and tibiae were dissected out, cut in half along their length, and immersed in the same fixative overnight. The samples were dehydrated in graded ethanol and then embedded in resin that was polymerized at 58C for 48 hr, as described previously (
For light microscopy, semithin sections (1 µm) of the primary spongiosa were cut with a glass knife on a Reichert Ultracut E microtome and stained with toluidine blue or by the von Kossa method. Thin sections of regions near the center of the growth plate were then prepared with a diamond knife, mounted on Formvarcarbon-coated nickel grids, and processed for postembedding protein A-gold immunocytochemistry (reviewed in
Immunolabeling for BSP and OPN
Grid-mounted tissue sections of LRW- and MMA-embedded undecalcified bone were floated for 15 min on a drop of 0.01 M PBS containing 1% ovalbumin (Oval; Sigma Chemical, St Louis, MO) to saturate nonspecific binding sites, and then were transferred to and incubated for 3 hr on a drop of polyclonal chicken egg yolk anti-rat OPN antibody (
Quantitative Analysis of the Immunolabeling
Images were printed at a final magnification of x38,000 and digitized using a Sony CCD 7002P camera (Sony Corporation; Tokyo, Japan). The number of gold particles per print, over interfibrillar accumulations of noncollagenous matrix (patches), and over collagenous regions of bone was quantified and compared using a Q500 image analysis system (Leica; Cambridge, UK). Based on the fact that NCPs accumulate mainly as interfibrillar patches (reviewed in
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Results |
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Structural Features
Von Kossa staining did not reveal any difference in the staining pattern and intensity observed over bone and calcified cartilage embedded in MMA or LRW (Fig 1A and Fig 1B). Ultrastructural preservation was generally better and more consistent with LRW processing, but MMA processing also permitted visualization of cellular features in some regions (compare Fig 2A and Fig 2B). Likewise, there was no significant difference in the appearance of the matrix (compare Fig 3A and Fig 3B, and Fig 3C and Fig 3D).
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Immunolocalization of BSP and OPN
The distribution of immunolabeling for BSP and OPN was similar for MMA- and LRW-embedded specimens. With both resins, gold particles were found mainly over the calcified bone matrix; some were also seen over osteoid. The labeling was mostly associated with electron-dense portions of the matrix, i.e., over cement lines and interfibrillar accumulations (patches) of granular/reticular-textured organic matrix (Fig 3). Incubation of tissue sections with protein Agold alone resulted in the occasional presence of particles throughout sections.
Quantitative Analysis of the Immunolabeling
Numerical parameters for the number of gold particles obtained after incubation of tissue sections with BSP and OPN antibodies are summarized in Table 1. For both BSP and OPN incubations, the distribution of patches/surface area within the three arbitrarily defined density categories was comparable for bone samples embedded in either MMA or LRW. For both resins, about 44% of the bone surface examined fell within the 816 patches/20 µm2 category and nearly 28% in the >17 patches category (Fig 4). With both antibodies, there was no significant difference in mean numbers of total gold particles/micrograph observed over the two resins (Fig 5A). Power analysis indicated that the number of samples studied was sufficient to allow an 80% chance of detecting a difference of 102.2 particles for BSP and 109.6 particles for OPN at a 0.05 level of statistical significance.
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The majority of the immunolabeling was found over the patches, and there was no significant difference in labeling density over them between the two resins (Fig 5B). Patches represented an average of 19% of the observed bone surfaces but an average of 88% of the labeling was found over them (Table 1). The relationship between labeling density and the number of patches was linear for both BSP and OPN (Fig 6).
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The mean number per area of gold particles obtained with both BSP and OPN over osteoid was 4.3 for MMA and 6.25 for LRW, over nuclei 2.5 and 3.5, respectively, and over cartilage 0 and 0.44, respectively (data not shown).
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Discussion |
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Whereas MMA is widely used for histomorphometric analysis of bone biopsy specimens, (
The labeling for BSP and OPN, respectively, obtained with undecalcified sections of MMA- and LRW-embedded bone (fixed as conventionally used with these resins) shows similar distributions and densities. In addition, the BSP:OPN labeling density ratios are essentially similar, indicating that MMA and LRW affect the antigenic properties of both BSP and OPN in a similar manner.
Matrix organization and relative proportions of collagenous and noncollagenous components in bone represent important parameters that vary with development stage and anatomic site, and reflect local tissue dynamics (
Although the number of gold particles over cartilage was almost nonexistent with both resins, osteoid showed roughly twice as many particles as over nuclei. Labeling over nuclei is used as an indication of background (
In conclusion, our study shows that it is possible to detect and immunolocalize BSP and OPN in sections of alcohol-fixed rat bone embedded in MMA from which the resin is not removed. This finding is important because it highlights the feasibility of processing experimental and clinical bone samples for parallel light microscopic and histomorphometric analyses, and for compositional studies at the ultrastructural level. In addition, it offers the possibility to further exploit valuable MMA-embedded bone samples from previous studies by determining whether the morphological and histomorphometric alterations observed are associated with changes in the content and/or distribution of major matrix proteins.
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Acknowledgments |
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Supported by the Canadian Institutes of Health Research.
We are grateful to Natalie Dion (Hôpital St Luc, Montréal) for MMA embedding, Sylvia Zalzal (Université de Montréal) for advice with immunolabeling, Micheline Fortin (Université de Montréal) for assistance with tissue sectioning, Pierre Rompré (Université de Montreal) for statistical analysis, and Paul Pilet (Inserm 99-03, Nantes) for adapting the gold particle counting software.
Received for publication June 5, 2002; accepted September 18, 2002.
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