RAPID COMMUNICATION |
Microwave Irradiation of Ethanol-fixed Bone Improves Preservation, Reduces Processing Time, and Allows Both Light and Electron Microscopy on the Same Sample
Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Québec, Canada (OL,VA-C,AN), and Metabolic Bone Diseases Laboratory, Centre de Recherche du CHUM, Hôpital Saint-Luc, Montréal, Québec, Canada (ND, L-GS-M)
Correspondence to: Dr Antonio Nanci, Faculty of Dentistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montreal, QC, Canada H3C 3J7. E-mail: antonio.nanci{at}umontreal.ca
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Summary |
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Key Words: microwave irradiation methylmethacrylate bone histomorphometry histochemistry immunolabeling ultrastructure
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Introduction |
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Electron microscopy has led to a more-detailed understanding of cell and tissue organization and of structure-function relationships. Furthermore, it is an important tool for the diagnosis of diseases difficult to clarify with the light microscope (Djaldetti et al. 1987). Ultrastructural analysis of bone biopsies allows a better definition of pathological elements and better understanding of their histogenesis, and has played a determining role in the characterization of a variety of bone-related neoplasms (Walaas and Kindblom 1990
; Goh et al. 2001
; Marucci et al. 2002
; Suh et al. 2002
).
MMA embedding of calcified or soft tissues has mostly been used for analyses at the light microscope level; its application in electron microscopy is not well documented (Baskin et al. 1992). In a recent report, we have shown that MMA is suitable for the ultrastructural immunolabeling of non-collagenous bone matrix proteins but that conventional ethanol fixation, widely used for histological procedures in the laboratory and hospital settings, yields inconsistent and, at best, suboptimal cellular preservation (Laboux et al. 2003
). Microwave irradiation (MWI) facilitates the penetration of fixatives and improves antigen detection (discussed in Arana-Chavez and Nanci 2001
). Therefore, we applied this approach to optimize preservation by ethanol and verified whether irradiation affects histological reactions for which MMA is conventionally used. The results demonstrate that MWI significantly reduces the processing time of calcified bone samples, improves preservation, and permits fluorochrome labeling for dynamic histomorphometry as well as a variety of light and electron microscope analyses.
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Materials and Methods |
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Procedures for animal handling were approved by the "Comité de Déontologie de l'Université de Montréal."
Human Bone Biopsy
Two contiguous transiliac crest biopsies were obtained from a 75-year-old osteoporotic male patient who had received tetracycline for dynamic histomorphometry. The biopsies were placed in 70% ethanol within minutes after their removal. About 1 hr later, one of them was subjected to three 5-min MWI cycles on ice, at 100% setting of the oven and the temperature programmed to a maximum of 37C. The specimen was immediately dehydrated in 80%, 90%, and three 100% changes of ethanol for periods of 30 min, with 5 min MWI on ice at each change. The other biopsy was kept in 70% ethanol for 1 week and then conventionally dehydrated and embedded in MMA as above. Procedure and patient consent forms for the biopsies were approved by the Ethics Committee of Hôpital St. Luc.
Bone Histomorphometry
Undecalcified MMA-embedded rat tibiae from conventionally fixed (n=4) and MWI-treated (n=4) samples and the two human transiliac biopsies were sectioned longitudinally with a Leica Polycut-E horizontal microtome. Five- and 8-µm-thick sections were cut at 60-µm intervals and mounted on gelatin-coated glass slides. The 5-µm-thick sections were deplasticized with toluene, rehydrated with decreasing concentrations of ethanol, and stained according to the Goldner's trichrome protocol. The 8-µm-thick sections were not deplasticized and were visualized unstained by epifluorescence for localization of tetracycline labeling.
Structural and static parameters of cancellous bone remodeling were quantified at the secondary spongiosa of the proximal metaphysis. Dynamic bone formation measurements were carried out at the extremity of the epiphysieal region. This region was selected to avoid autofluorescence associated with cartilage and weakly mineralized bone that is frequently observed in metaphyseal trabecular bone in young growing rats. Histomorphometry was also carried out on the cancellous bone of the human biopsies. Measurements were performed with a semiautomatic image analyzing system consisting of a Leica Polyvar light microscope equipped with a camera lucida and digitizing tablet linked to a computer. The data were acquired and analyzed using the OsteoMeasure software (Osteometrics; Decatur, GA). Nomenclature and abbreviations of histomorphometric parameters follow the recommendations of the American Society for Bone and Mineral Research (Parfitt et al. 1987).
Histochemistry
Detection of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRACP) activity was carried out on 5-µm-thick sections according to the method of Liu et al. (1987). Naphthol-AS-TR was used as substrate for both enzymes, and Fast Blue BB salt (Sigma-Aldrich) and pararosaniline were used as couplers for ALP and TRACP, respectively.
Immunohistochemistry
Five-µm-thick sections were deplasticized with toluene and rehydrated through decreasing concentrations of ethanol to distilled water. Sections were first treated with peroxidase blocking reagent from the DAKO EnVision+ System, horseradish peroxidase (HRP) (DAB) Kit (DAKO; Carpinteria, CA), blocked for 20 min with 5% powdered skimmed milk in 0.01 M phosphate buffered saline (PBS), and then incubated for 1 hr with polyclonal rabbit anti-osteopontin (OPN) antibody (LF-123; courtesy of L.W. Fisher, NIDCR, Bethesda, MD) or with anti-bone sialoprotein (BSP) antibody (LF-100; courtesy of L.W. Fisher). Sections were then washed in 0.01 M PBS containing 0.01% Tween-20, and incubated for 30 min with labeled polymer, HRP anti-rabbit antibody (DAKO EnVision). The sections were then washed with PBSTween followed by distilled water, treated with DAB chromogen (DAKO EnVision) for 6 min, and then counterstained with methyl green. Finally, the sections were dehydrated in ethanol, passed in xylene, and covered with a glass coverslip mounted with Permount (Fisher Scientific; Fair Lawn, NJ). Controls consisted of omitting the primary antibody and incubation with a variety of unrelated primary antibodies. All incubations were carried out in a moist environment at room temperature.
Processing for Light and Transmission Electron Microscopy
Semithin sections (1 µm) for light microscopy were cut with a diamond histoknife on a Reichert Ultracut E microtome and stained with toluidine blue. Some sections were processed for mineral detection by the von Kossa method and lightly counterstained with toluidine blue. For ultrastructural observations, thin sections (100 nm) were cut with a diamond knife and mounted on Formvarcarbon-coated nickel grids. All thin sections used for morphology or immunolabeling were stained with uranyl acetate and lead citrate before examination with a JEOL JEM 1200EX-II operated at 60 kV.
Postembedding Colloidal Gold Immunolabeling
Grid-mounted sections were processed for postembedding protein Agold immunolabeling (reviewed in Bendayan 1995). Briefly, sections were floated for 15 min on a drop of 0.01 M PBS, pH 7.2, containing 1% ovalbumin (Oval) (Sigma-Aldrich) to saturate nonspecific binding sites, and then transferred to and incubated for 1 hr on a drop of a chicken egg yolk anti-rat OPN antibody (diluted 1:50 in PBS; Nanci et al. 1996
). Sections were then rinsed with PBS, blocked with PBSOval for 15 min, and incubated with a rabbit anti-chicken gold-conjugated secondary antibody (Cappel Research Products; Scarborough, ON, Canada) diluted 1:5 with PBS, pH 8. As controls, sections were incubated with the secondary antibody only. All procedures were performed at room temperature. After the secondary antibody, grids were washed thoroughly with PBS, rinsed with distilled water, and air dried.
Backscattered Electron Imaging
Tissue blocks faced for cutting sections were directly examined using a backscattered electron detector in a JEOL JSM-LV 6460 variable pressure scanning electron microscope operated at 15 kV and a pressure of 2550 Pa.
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Results |
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Discussion |
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One major problem with fixation by immersion is that the quality of preservation depends on the speed at which the fixative penetrates the sample. The centers of samples, particularly larger ones such as biopsy specimens, are usually less well preserved. MWI facilitates and accelerates the penetration of fixatives, in our case ethanol, into samples, reducing the diffusion rate effect on preservation (Wagenaar et al. 1993; Massa and Arana-Chavez 2000
). In addition, it has been proposed that MWI stabilizes tissue proteins, but as yet it is not clear whether any crosslinking is induced in the presence of ethanol or whether the stabilizing effect observed simply results from temperature-induced protein coagulation (Moran et al. 1988
; discussed in Visser et al. 1992
). In any case, our results demonstrate that tissue preservation is improved by MWI of ethanol-immersed bone samples. The resulting structural details are comparable to those that are usually obtained when tissues are simply immersed in mild fixatives and then embedded in acrylic resins.
Clearly, for both experimental and clinical analyses, it would be very useful to cross-match information obtained by various techniques from the same sample. Transgenic/knockout animals are proving to be very powerful models for understanding the function of proteins and how alteration in their production may, directly or indirectly, lead to disease. Such animal models are complex to produce and maintain, and hence are limited in availability. Any methodology that allows extraction of maximal information from precious tissue samples will certainly help improve our understanding of the function of gene products and phenomena that lead to pathological alterations.
Characterization of the biochemical profile of bones and variations induced by pathological alterations is complicated by the fact that this profile varies according to the anatomic location and also at the level of the microenvironment (discussed in Nanci 1999). The organic matrix of bone consists mainly of collagen type I and small amounts (
10%) of several non-collagenous matrix proteins, which play major roles in cellular and extracellular events (reviewed in Boskey 1996
; Gehron Robey 1996
). Disease-induced variations in the minor non-collagenous components would be difficult to determine biochemically. Immunocytochemistry is a form of "biochemistry on section" that allows detection of small quantities of a constituent and its localization with respect to others at the site where they accumulate and likely express their action, in normal and diseased bone. Furthermore, the localization of matrix proteins at the ultrastructural level is important for determining their relationship with osteoblasts and osteoclasts and with sites of mineralization events. The fact that sections of MMA-embedded tissues can be deacrylated is likely to enhance the sensitivity of immunolabeling and the detection of low-abundance epitopes, such as membrane proteins or low-level expression gene products. It also permits investigation of mRNA expression by in situ hybridization (Saito et al. 1999
). Because the ethanol-MMA combination preserves mRNAs, deacrylated MMA sections of ethanol-fixed tissues could, in theory, also be used for in situ RT-PCR and mRNA extraction from laser-dissected samples. Normally, laser dissection is carried out on paraffin-embedded tissues. In the case of calcified tissues, this requires an additional decalcification step, which may result in the loss and/or deterioration of the mRNA. On the other hand, MMA-embedded calcified tissues can readily be cut at various thicknesses and, after deacrylation, would be amenable to mRNA detection and extraction methodologies. The ability to carry out all the above evaluations on the same specimen offers the advantage of correlating macroscopic data, bone formation and resorption parameters, molecular markers of cell activity, and mineral status and composition of the organic matrix.
In summary, in this study, we suggest a protocol for processing calcified bone samples, which essentially consists of treating with MWI samples conventionally immersed in 70% ethanol. This simple protocol requires only minor adaptations to current laboratory procedures and significantly shortens tissue-processing time. It broadens the spectrum of analytical methodologies that can be applied to the same sample and allows more structural and compositional information to be obtained. This enhanced approach makes it possible to fully achieve the concept of "molecular histomorphometry" expounded by Parfitt (1994), which is the integration of conventional histomorphometry with molecular information to provide new insights into the physiology and pathology of bone.
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Acknowledgments |
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We are grateful to Sylvia Francis Zalzal (Université de Montréal) for her help with backscattered electron imaging, to Micheline Fortin (Université de Montréal) for assistance with tissue sectioning and immunolabeling, and to Roxane Carrier and Claire Deschêsnes (Hôpital St Luc) for technical participation in histomorphometric preparations. O.L. was the recipient of an INSERM/Canadian Institutes of Health Research award (International Scientific Exchanges IRSC/INSERM; XIF-40990).
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Footnotes |
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2 Present address: Laboratory of Mineralized Tissue Biology, Department of Histology and Embryology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
Received for publication May 30, 2004; accepted June 1, 2004
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