New embedding medium for sectioning undecalcified bone.

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Quantitative immunogold labeling of bone sialoprotein and osteopontin in methylmethacrylate-embedded rat bone.

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 A-gold 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.     

Microwave irradiation of ethanol-fixed bone improves preservation, reduces processing time, and allows both light and electron microscopy on the same sample.

Methylmethacrylate (MMA) embedding is routinely used for histomorphometry of undecalcified bone preserved by prolonged immersion in ethanol, a procedure that yields poor ultrastructural detail. Because microwave irradiation (MWI) facilitates penetration of fixatives, we have investigated whether it can improve preservation by ethanol. Rat tibiae, some labeled with tetracycline, and a human iliac crest biopsy were immersed in 70% ethanol and dehydrated, both under MWI, for a total processing time of approximately 7 hr. Controls were not irradiated, and all specimens were embedded in MMA at 4C. They were then processed for histomorphometry, histochemistry, structural analysis, and immunolabeling. The results showed that histological preservation was improved with MWI. Static bone formation and resorption parameters and rate of mineral apposition were similar to those of conventionally processed specimens. Mineral distribution, as visualized by von Kossa staining and backscattered electron imaging, was not affected. Alkaline phosphatase and tartrate-resistant acid phosphatase activity, as well as immunolocalization of bone sialoprotein and osteopontin, were readily visualized. Ultrastructurally, osteopontin exhibited a typical distribution in mineralization foci, between calcified collagen fibrils, and at cement lines. These data show that MWI improves preservation and permits application of a broad spectrum of analytical methodologies on the same bone sample while considerably reducing processing time.     

Embedding and Sectioning Undecalcified Bone, and its Application to Radioautography

A method is described for embedding and sectioning hard, undecalcified bone, which is designed for use by technical personnel. Bone fixed in a variety of ways is progressed through alcohols to ether-alcohol and then infiltrated with ether-alcohol solvented plastic (plasticized nitrocellulose) by a combination of centrifugation and high pressure embedding technics. The ether-alcohol is evaporated in a partially closed container in a manner similar to that employed in celloidin embedding, but differs from the latter by the removal of all of the solvent. Celloidin is the source of nitrocellulose and Amoil-S, the added plasticizer. Undecalcified adult bone of all types is readily cut at a thickness of 5-8μ on a heavy duty sliding microtome. The sections are then mounted on gelatinized slides. The procedures for preparing strip film radio-autograms of bone sections and subsequent staining of the preparation are given. The results obtained are illustrated.     

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.     

Evaluation of LR white resin for histology of the undecalcified rat tibia

Histology of plastic embedded undecalcified bone represents a challenging problem to the histotechnologist. We outline here an exploration of LR White resin as a suitable medium for histologic study of undecalcified rat tibia. A procedure was developed for light microscopy of rat tibia embedded in LR White and sectioned by sawing-grinding technics. The specimens were fixed in 10% neutral buffered formalin or alcohol-acetic acid-formol, dehydrated in ethanol, defatted in chloroform followed by resin infiltration and heat-curing of embedded blocks. The procedure of dehydration, defatting, infiltration, and polymerization can be completed within 10 days. Cold curing with accelerator provided by the manufacturer did not yield superior results compared to blocks cured with heat. Thick sections were obtained using a diamond wire saw, attached to plexiform slides, then ground and polished. Surface staining with Von Kossa silver reagent or toluidine blue revealed satisfactory morphological preservation of the mineralized bone sections. Artifacts like small bubbles appeared occasionally and could not be avoided despite prolonged infiltration or cold curing of blocks. Our method is relatively simple for base-line histologic study of rat tibia. The method offers advantages such as easy adaptability, reliable stainability, contrast, and resolution of bone architecture and marrow cells. Two other embedding media, Micro-Bed resin and Unicryl, were also tested, but produced inferior results.     

Serial Sections from Plastic-Embedded Specimens; Arthropods in Methacrylate

Testing of embedding technics adapted to 1-5 μ sectioning of mites in their developmental stages has lead to the selection of methacrylate resins. This kind of embedding medium supplies the bonding necessary, especially with yolky eggs, to maintain the integrity of fragile or refractory specimens during sectioning. Whereas paraffin offers the advantage of ribboning, the customary use of a plastic matrix does not. However, DeGiusti and Ezman (1955) use mixtures of plastic and paraffin to obtain the ribboning qualities of the latter.     

Cement Line Staining in Undecalcified Thin Sections of Cortical Bone

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 μm, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

A New Method for Identification of Cement Lines in Undecalcified, Plastic Embedded Sections of Bone

A gallocyanin method for demonstrating cement lines in thin, undecalcified sections of bone has been developed that is compatible with prestaining with ostcochrome before plastic embedding. After sectioning at 5 pm on the Jung K heavy duty microtome, the sections are attached to a microslide using Haupt's adhesive mounting medium, placed on a slide warmer at 37 C until completely dry, and deplasticized in xylene at 45 C for 16-44 hr. Sections are stained with 0.15% gallocyanin-5% chrome alum solution for 30 min, followed by staining in buffered Villanueva blood stain for 1-1 1/2 hr, quickly dehydrated, differentiated in equal parts xylene and 100% ethanol, cleared, and mounted in Eukitt's medium. Reversal lines appear as thin, scalloped, blue or purple lines approximately 0.3 pm wide, and arrest lines as thick, homogeneous, straight or evenly curved, dark blue or purple lines approximately 2 pm wide. The method also demonstrates abnormal halo volumes around ostcocytes, old and new bone matrix, osteoid seams, and the granular mineralization front at the osteoid-bone interface. It promises to be valuable in the study of age-related bone loss, osteoporosis, and metabolic bone disease.     

A new method for identification of cement lines in undecalcified, plastic embedded sections of bone

A gallocyanin method for demonstrating cement lines in thin, undecalcified sections of bone has been developed that is compatible with prestaining with osteochrome before plastic embedding. After sectioning at 5 microns on the Jung K heavy duty microtome, the sections are attached to a microslide using Haupt's adhesive mounting medium, placed on a slide warmer at 37 C until completely dry, and deplasticized in xylene at 45 C for 16-24 hr. Sections are stained with 0.15% gallocyanin-5% chrome alum solution for 30 min, followed by staining in buffered Villanueva blood stain for 1-1 1/2 hr, quickly dehydrated, differentiated in equal parts xylene and 100% ethanol, cleared, and mounted in Eukitt's medium. Reversal lines appear as thin, scalloped, blue or purple lines approximately 0.3 micron wide, and arrest lines as thick, homogeneous, straight or evenly curved, dark blue or purple lines approximately 2 microns wide. The method also demonstrates abnormal halo volumes around osteocytes, old and new bone matrix, osteoid seams, and the granular mineralization front at the osteoid-bone interface. It promises to be valuable in the study of age-related bone loss, osteoporosis, and metabolic bone disease.     

Cement line staining in undecalcified thin sections of cortical bone.

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Better Epoxy Resin Embedding for Electron Microscopy at Low Relative Humidity

In the absence of other factors known to influence sectioning properties, high environmental relative humidity is shown to yield poorly embedded tissue. Humidity-related effects are avoided if the following embedding precedure is used: impregnate tissues using the following solutions 1) 70% alcohol—5 minutes, 2) 95% alcohol—4 × 15 minutes, 3) absolute alcohol—3 × 40 minutes, 4) acetone—2 × 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 814,39.4 g; DMP-30, 2.6 g)— 1 hour, 6) acetone-epoxy resin 13—1 hour, 7) epoxy resin—1 hour: complete the preparation of blocks as follows 8) when tissues have been oriented in epoxy resin in flat embedding molds, place molds in one evacuated vacuum desiccator 10 cm above a 2 cm layer of Drierite for 24 hours at room temperature, 9) raise temperature to 60 C and maintain for 3 days to cure resin.     

An Embedding Method for Histochemical Studies of Undecalcified Skeletal Growth Plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tissues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

An embedding method for histochemical studies of undecalcified skeletal growth plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tisssues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

Glycol methacrylate as an embedding medium for bone

A simple and reliable procedure for embedding undecalcified trabecular bone tissue in noncommercial glycol methacrylate (GMA) has been developed. The embedding mixture includes a monomer, methacrylic acid hydroxyethyl ester; a copolymer, methacrylic acid butyl ester; a cross-linker, ethylene glycol dimethacrylate; a catalyst, Luperco; a chemical initiator (N,N-dimethylaniline) and, to avoid excessive elevation of temperature during polymerization, a heat moderator, alpha-terpinene. The appropriate proportions of these components have been selected to give specimens which can be easily sectioned with classical microtomes and which do not swell but spread evenly on a water surface. Since polymerization occurs at -4 C, the method allows demonstration of such enzymatic activities as acid and alkaline phosphatase and carbonic anhydrase. It provides excellent preservation of bone tissue and in studies of bone metabolism allows histomorphometry as well as visualization of fluorescent labeling and radioactive markers. The cost is significantly less than available commercial kits. In our hands glycol methacrylate is at present more useful than methyl methacrylate and is used in our laboratory for routine embedding of bone tissue.     

Orientation of Small Specimens for Cryosectioning

A simple technique is introduced to achieve symmetrically oriented frozen sections of small specimens such as young fish or frog larvae. Small samples are especially difficult to orient if they are already frozen to the chuck in a freezing microtome. Orientation of the sample in a mold filled with embedding medium prior to freezing permits sectioning as well as easy labeling and storage of the specimens. The use of a stereo microscope during orientation is optional.     

Embedding Thin Plant Specimens for Oriented Sectioning

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.     

A Versatile New Mineralized Bone Stain for Simultaneous Assessment of Tetracycline and Osteoid Seams

A versatile mineralized bone stain (MIBS) for demonstrating osteoid seams and tetracycline fluorescence simultaneously in thin or thick undecalcified sections has been developed. Bone specimens are fixed in 70% ethanol, but 10% buffered formalin is permissible. Depending upon one's preference, these specimens can be left unstained or be prestained before plastic embedding. Osteoid seams are stained green to jade green, or light to dark purple. Mineralized bone matrix is unstained or green. Osteoblast and osteoclast nuclei are light to dark purple, cytoplasm varies from slightly gray to pink. The identification of osteoid seams by this method agrees closely with identification by in vivo tetracycline uptake using the same section from the same biopsy. The method demonstrates halo volumes, an abnormal, lacunar, low density bone around viable osteocytes in purple. This phenomenon is commonly seen in vitamin D-resistant rickets, fluorosis, renal osteodystrophy, hyperparathyroidism, and is sometimes seen in fluoride treated osteoporotic patients. In osteomalacic bone, most osteoid seams are irregularly stained as indicated by the presence of unmineralized osteoid between mineralized lamellae. The method has been used effectively in staining new bone formation in hydroxyapatite implants and bone grafts. Old, unstained, plastic embedded undecalcified sections are stained as well as fresh sections after removal of the coverslip. This stain also promises to be valuable in the study of different metabolic bone diseases from the point of view of remodeling, histomorphometry, and pathology.