TECHNICAL NOTE |
Correspondence to: Reinhold G. Erben, Inst. of Physiology, Physiological Chemistry, and Animal Nutrition, U. of Munich, Veterinärstr. 13, 80539 Munich, Germany.
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Summary |
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Methylmethacrylate (MMA) embedding of undecalcified bone biopsies is a technique widely used for bone histomorphometry. However, conventional MMA embedding causes almost complete loss of enzyme activity and protein antigenicity in the tissues. Recently, an MMA embedding technique has been reported that preserves enzyme activity and antigenic determinants in bone tissue. We describe here a modification of this embedding method. For our modified MMA embedding process, commercially available methacrylates can be used without purification, and the histologic quality of bone sections is comparable to that of conventionally MMA-embedded bone specimens. The technique reported here can be employed for embedding of larger bone samples and is suitable for histochemical and immunohistological applications as well as for routine bone histomorphometry. By addition of methylbenzoate during infiltration and polymerization of the plastic, the antigenicity of the tissue was improved. As applications of this novel technique, demonstration of alkaline phosphatase and tartrate-resistant acid phosphatase as well as positive labeling of Kupffer cells and osteoclasts with the monoclonal antibody ED1 in sections of liver, tibiae, and vertebrae of 3-month-old rats was demonstrated. The method described here might be useful for the inclusion of histochemical and immunohistological methods into bone histomorphometry. (J Histochem Cytochem 45:307-313, 1997)
Key Words: Methylmethacrylate, Methacrylates, Bone, Rats, Histomorphometry, Histochemistry, Immunohistochemistry, Methylbenzoate
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Introduction |
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Methylmethacrylate (MMA) embedding of undecalcified bone biopsies is a technique widely used for bone histomorphometry and bone marrow hematopathology, both in clinical medicine and in animal experimental studies (
Polymerization of methacrylates is an exothermic radical chain reaction. Conventional MMA mixtures mostly use organic peroxides as initiators of polymerization, and polymerization is usually carried out at temperatures between 30 and 45C, using thermal decomposition of the peroxide to start the polymerization process (
In vivo labeling of bone with fluorochromes (e.g., tetracyclines) is a widely used method to assess mineral apposition rate and bone formation rate in bone histomorphometry. Fluorochromes are calcium-seeking molecules that bind to the mineralization fronts in bone formation sites. In sections of undecalcified bone, fluorochromes can be visualized by their specific fluorescence under UV or blue light excitation. Prolonged exposure to blue or UV light results in irreversible fading of fluorochromes bound to bone mineralization sites. Therefore, possible fading of fluorochromes precludes the use of light for polymerization of the methacrylate in a routine plastic embedding of undecalcified bones suitable for both bone histomorphometry and histochemical and immunohistological techniques.
Recently, an MMA embedding technique has been reported that can be used for embedding of larger bone samples, and which preserves enzyme activities and antigenic determinants in bone tissue (
We report here a modification of the embedding technique originally described by
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Materials and Methods |
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Animal Procedures
Six 3-month-old female Fischer-344 rats (Charles River; Sulzfeld, Germany) were used for this study. The rats were housed in pairs at 24C and a 12-hr/12-hr light/dark cycle with free access to tapwater and a commercial rat diet (Altromin 1320; Altromin, Lage, Germany). On Days 14, 9, and 4 before sacrifice, each rat was injected IP with the fluorochromes demeclocycline (20 mg/kg), alizarin complexone (30 mg/kg), and calcein (20 mg/kg), respectively. All fluorochromes were purchased from Sigma (Deisenhofen, Germany). After this labeling protocol, the rats were sacrificed by an ether overdose, and parts of the liver, the proximal tibiae, and the lumbar vertebrae were removed. The bones were carefully defleshed and the marrow cavity in the tibiae was opened by removing a small part of the lateral condyle, and in the vertebrae by cutting off the intervertebral discs. The experimental protocol was approved by the local government authorities.
Histology
If not otherwise specified, all chemicals were purchased from Merck (Darmstadt, Germany). All steps of fixation, dehydration, and infiltration were carried out at 4C on a magnetic stirrer. The tissue samples were fixed in 40% ethanol for 48 hrs or in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, at pH 7.4, for 24 hr. The tissue specimens fixed in PFA were washed overnight in 0.1 M phosphate buffer, pH 7.4, containing 10% sucrose. Subsequently, the bone and liver samples were dehydrated according to the following schedule (
After trimming of the plastic blocks, 3-5-µm thick sections were prepared at room temperature with a Microm HM 360 microtome (Microm; Walldorf, Germany) equipped with a D profile knife with a tungsten carbide cutting edge. During sectioning, the knife and the blocks were kept moist with a sectioning fluid (WIV; Schwetzingen, Germany) or with 30-40% ethanol. The sections were transferred onto chromalum-gelatin-coated slides (for conventional staining and fluorochromes) or to slides pre-treated with 3-aminopropyltriethoxy-silane (APES, Sigma; for histochemistry and immunohistochemistry) and carefully stretched using 70% ethanol. Thereafter, the sections were covered with a polyethylene foil, flattened with a rubber roller, pressed with a slide press, and dried for 2 days at 42C in the slide press.
For deplasticization, the sections were placed in three changes of 2-methoxyethylacetate for 20 min each, two changes of acetone for 5 min each, and two changes of deionized water for 5 min each. Sections were routinely stained with toluidine blue at acid pH (
Histochemistry
For demonstration of AP activity, deplasticized sections were transferred into 0.1 M Tris-HCl buffer, pH 8.2, for 5 min, and the histochemical reaction was then carried out in 0.1 M Tris-HCl buffer, pH 8.2, using a commercially available kit (Vector Red; Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Positive staining developed within 30 min to 2 hr at 37C. Control sections were incubated in incubation medium without the enzyme substrate. No staining developed in these control sections.
For TRAP histochemistry, deplasticized sections were placed in 0.1 M acetate buffer, at pH 5.0, for 5 min, and the TRAP reaction was subsequently performed as described previously by
Counterstaining for sections stained for AP and TRAP was performed using Mayer's hematoxylin (1 min), and the sections were subsequently mounted with an aqueous mounting medium (Kaiser's glycerol gelatin; Merck).
Immunohistochemistry
Immunohistochemistry was performed using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method. Deplasticized sections were placed in 0.1 M Tris-HCl buffer, at pH 8.2, for 5 min. After blocking the sections with 10% rabbit serum (Vector) for 20 min, the sections were incubated overnight at 4C with the MAb ED1 (Serotec; Oxford, UK) at a 1:100 dilution in Tris-buffered saline (TBS; 0.1 M Tris, 0.15 M NaCl, pH 8.2) with 1% rabbit serum. Control sections were incubated with a non-immune myeloma IgG1 (clone MOPC-21; Sigma) at the same concentration as the ED1 antibody. After washing in TBS containing 0.1% Tween 20 (three times for 5 min), the sections were incubated for 30 min with rabbit anti-mouse antibody (STAR 37, not crossreacting with rat; Serotec) at 1:25 dilution in TBS with 1% rabbit serum. After washing (three times for 5 min in TBS with 0.1% Tween 20), sections were incubated for 30 min with mouse monoclonal APAAP complex (Dako; Hamburg, Germany) at 1:50 dilution in TBS. Thereafter, the sections were rinsed (three times for 5 min in TBS with 0.1% Tween 20), and stained with the Vector Red kit according to the supplier's instructions. Levamisole (Vector) was added to the incubation medium at the concentration recommended by the supplier. Finally, the sections were counterstained with Mayer's hematoxylin for 1 min and mounted with an aqueous mounting medium (Kaiser's glycerol gelatin).
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Results |
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The embedding protocol employed in this study resulted in reliable and homogeneous polymerization of the blocks. The tendency to form bubbles during polymerization at the interface between the glass vial and the tissue surface was reduced when glass vials with a pre-polymerized base were used. Hardness of the blocks, sectioning properties, stretching of the sections, and section quality were comparable to those of conventionally MMA-embedded bone specimens. The hardness of the blocks did not increase even after prolonged storage at room temperature (months to years). The staining characteristics of the sections were excellent. The staining protocols used for sections of conventionally MMA-embedded bones could be used without any modification. Compared to fixation with 40% ethanol, PFA fixation resulted in better morphological detail, with little shrinkage of bone marrow cells (Figure 1). In vivo fluorochrome labeling of the bones could be visualized by fluorescence microscopy. The intensity of the fluorescence signals was independent of the fixation used and was comparable to that of specimens embedded in conventional MMA (Figure 2).
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Osteoclasts on all periosteal and endosteal bone surfaces stained positive for TRAP activity (Figure 3). TRAP-positive cells not in direct contact or in close proximity to bone surfaces occurred only very rarely in bone marrow. The intensity of TRAP activity was about the same in 40% ethanol- and PFA-fixed specimens. AP activity, on the other hand, was more sensitive to different fixation processes and was greatly reduced by PFA fixation. Osteoblasts as well as many leukocytes in bone marrow stained positive for AP in specimens fixed with 40% ethanol at 4C (Figure 4). Cells positive for AP showed a membranous staining pattern in most cases. For both TRAP and AP, staining was absent in control sections incubated in medium lacking the enzyme substrate.
When sections of rat liver were exposed to MAb ED1, Kupffer cells in liver sinuses were intensely labeled (Figure 5A). The liver sections were used as positive controls for ED1 labeling. In bone tissue, osteoclasts on periosteal (Figure 5B) and endosteal bone surfaces, as well as osteoclasts (chondroclasts) located beneath the growth plate (Figure 5C), stained strongly positive with MAb ED1. There were also many ED1-positive cells in bone marrow. All cells reactive to ED1 showed a typical cytoplasmic staining pattern (Figure 5A-C). Reactivity to MAb ED1 in liver and bone tissue was observed with about the same intensity for both ethanol- and PFA-fixed specimens. An isotype-specific non-immune control MAb used at the same concentration as the ED1 antibody did not result in positive staining. At the dilution of primary antibody used in this study, nonspecific background staining was almost absent in sections of liver and bone tissue. Addition of levamisole at the concentration recommended by the manufacturer completely inhibited endogenous AP activity in liver, bone marrow leukocytes, and osteoblasts in most specimens. In ethanol-fixed tissue it was sometimes necessary to double the concentration of levamisole in the incubation medium to inhibit endogenous AP activity.
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Discussion |
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Based on the MMA embedding technique described by
High-quality undecalcified sections with few or no artifacts are a prerequisite for reliable histomorphometric measurements (
The enzyme activities of AP and TRAP were well preserved by the embedding technique used in this study. TRAP is a lysosomal enzyme expressed at high levels in committed osteoclast precursors and mature osteoclasts (
With the methods employed in this study, Kupffer cells in the liver and osteoclasts were intensely labeled by the MAb ED1. Proteolytic digestion was not necessary for demonstration of positive staining with ED1. In our experience, the rat antigen reacting with MAb ED1 is resistant to most fixatives (e.g., ethanol, acetone, PFA, formalin, Schaffer's solution, and Carnoy's solution). The intense labeling of cells by immunohistological techniques shown in this study is likely due to the fact that complete removal of plastic is possible in sections of MMA-embedded tissue (
In summary, we have described a novel MMA embedding technique that can be used for standard bone histomorphometry and allows the application of histochemical and immunohistochemical methods. Therefore, this method meets the criteria for a more broad application in bone research, and might be useful for the inclusion of histochemical and immunohistological methods in bone histomorphometry. In combination with histochemical or immunohistological methods specific for osteoblasts or osteoclasts, this embedding method may also be useful as a basis for automatic detection of these cells in undecalcified sections of bone tissue by image analysis techniques. Moreover, the embedding technique described here may represent a further step towards the integration of different disciplines into bone research ["molecular histomorphometry" (
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Acknowledgments |
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I thank Stefanie Engert for expert technical assistance.
Received for publication June 14, 1996; accepted September 18, 1996.
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Literature Cited |
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