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.     

Two Methods for Flat Embedding Sections in LR White Cut from Paraffin Blocks

Two simple methods are described for flat embedding of sections cut from paraffin blocks of brain tissue in the hydrophilic resin LR White. The ability of fresh resin to polymerize when added to already cured resin is exploited. In the first method, a tissue section with a drop of LR White is pressed to the bottom of a gelatin capsule using a blank block. In the second method, the section in a drop of resin is sandwiched between the sawed halves of a blank block. After curing, thin sections containing large areas of tissue can be collected and processed for immunocytochemistr y.     

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.     

Use of LR white resin for post-embedding immunolabelling of brain tissue.

The object of this study was to investigate the applicability of the acrylic resin 'LR White' to immunolabelling of various antigenic determinants in aldehydefixed rat CNS tissue. Antibodies were used, which worked well in paraffin sections and therefore were suitable to detect antigens resistant to complete dehydration and heat. Different LR White embedding protocols were employed in order to select the preparation conditions that adequately preserved both the antigenicity and fine structure. Specimens were completely dehydrated with up to 100% ethanol, which was followed by various infiltration times with LR White monomer. Polymerization of the resin was induced by heat, a chemical catalytic procedure (accelerator), or ultraviolet (UV) light. Paraffin, as well as semithin and ultrathin LR White sections were incubated with antibodies reacting to antigens located on the cell surface (stage-specific embryonic antigen-1; SSEA-1), within the plasma membrane (myelin basic protein), in the cytosol (HNK-1, S100 protein), in the cytoskeleton (GFAP, vimentin, neurofilament protein, INT-FIL), and in the extracellular matrix (laminin). All of the examined antigens were immunocytochemically detectable in paraffin-embedded material, while the carbohydrate moieties, HNK-1 and SSEA-1, were not immunoreactive in LR White sections. However, in cryostat sections processed for pre-embedding immunoelectron microscopy, the HNK-1 epitope and SSEA-1 were immunolabelled. Polymerization carried out under UV light led to better structural preservation of brain tissue than resin cured with heat or catalyst. The length of prior infiltration with monomer apparently had no effect on tissue preservation. Consequently, UV light-induced polymerization of LR White gives acceptable morphology of brain tissue. However, the use of this acrylic resin is restricted to the detection of some CNS antigens only.     

Large area sectioning for morphologic studies of nonhuman primate nasal cavities

The nasal region is important for studies in inhalation toxicology but is difficult to prepare for histological examination, especially in species as large as primates. A method for the histologic preparation of undecalcified, complete transverse sections of the nonhuman primate nasal cavity is summarized as follows. After removal of excess soft tissue, mandible and calvaria, the head is fixed in 10% neutral buffered formalin. The nasal region is transversely sectioned into serial 3-mm-thick blocks from the nares to the posterior aspect of the soft palate using a low speed saw with a water-cooled diamond-coated blade. The blocks are embedded in a mixture of glycol and methyl methacrylates, with polyethylene glycol-1500 and dibutylphthalate as plasticizers. The plastic blocks average 5.0 x 5.0 x 1.5 cm; 2-4 microns sections are cut on an automated sliding microtome. In spite of the size of the blocks, this technique yields complete transverse sections of the nasal cavities with excellent morphologic detail. The sections are amenable to a wide range of staining procedures. The procedure lends itself to autoradiographic studies. The embedding mixture is ideally suited for studies of undecalcified bone and teeth.     

A Method for Histological Preparation of Undecalcified Bone Sections Containing Acrylic Bone Cement

An improved and time reducing method is presented for the histological evaluation of bone containing polymethylmethacrylate (PMMA) bone cement. The undecalcified bone was embedded in epoxy resin and sections of 50-100 μm thickness were produced using a commercially available cutting grinding system. The sections were stained with Stevenel's blue and van Gieson picrofuchsin or a modified hematoxylin-eosin. PMMA bone cement was not dissolved and remained enabling examination in situ of an intact cement bone interface and tissue reaction without decalcification.     

Histochemical studies of acid and alkaline phosphatases in rat tooth germs with undecalcified resin-embedded specimens.

A novel technique for the histochemical demonstration of acid phosphatase (AcPase) and alkaline phosphatase (AkPase) in hard tissues has been proposed. Fresh, unfixed, undecalcified samples of rat tooth germs and surrounding structures were embedded in LR Gold resin at -20 degrees C. Sections of 2 microns were taken and subsequently processed for enzyme histochemistry. AkPase reaction product appeared as strong linear staining outlining cell boundaries and was present in the enamel organ, dental pulp, and osteoblast cells. Tartrate-resistant AcPase staining was seen exclusively in the osteoclasts of developing alveolar bone. Our results demonstrated that the use of unfixed, undecalcified LR Gold resin-embedded specimens for histochemistry is a novel technique which may be of value for certain studies when decalcification of specimens is undesirable. The technique appears to give good preservation of enzyme activity combined with the ability to prepare sections with excellent morphological detail.     

Large Area Sectioning for Morphologic Studies of Nonhuman Primate Nasal Cavities

The nasal region is important for studies in inhalation toxicology but is difficult to prepare for histological examination, especially in species as large as primates. A method for the histologic preparation of undecalcified, complete transverse sections of the nonhuman primate nasal cavity is summarized as follows. After removal of excess soft tissue, mandible and calvaria, the head is fixed in 10% neutral buffered formalin. The nasal region is transversely sectioned into serial 3-mm-thick blocks from the nares to the posterior aspect of the soft palate using a low speed saw with a water-cooled diamond-coated Made. The blocks are embedded in a mixture of glycol and methyl methacrylates, with polyethylene glycol-1500 and dibutylphthalate as plasticizers. The plastic blocks average 5.0 × 5.0 × 5.1 cm; 2-4 μn sections are cut on an automated sliding microtome. In spite of the size of the blocks, this technique yields complete transverse sections of the nasal cavities with excellent morphologic detail. The sections are amenable to a wide range of staining procedures. The procedure lends itself to autoradiographic studies. The embedding mixture is ideally suited for studies of undecalcified bone and teeth.     

A Procedure for Preparing Undecalcified and Unembedded Bone Sections for Light Microscopy

We have developed a procedure for light microscopic investigation of undecalcified and unembeddedbone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 μm thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.     

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.     

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization,cellular and structural detail

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.     

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization, cellular and structural detail

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.     

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.     

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

Summary We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([3H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [3H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [3H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

Enhancement of the periodic acid--Schiff (PAS) and periodic acid--thiocarbohydrazide--silver proteinate (PA-TCH-SP) reaction in LR white sections

LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination: 1) The PAS reaction in semithin sections turns out stronger after partial (70% ethanol) than complete (100% ethanol) dehydration of the tissue before its transfer to 100% LR White. 2) Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end. 3) The use of hot silver proteinate (50 degrees C) plus strong silver enhancement (15-20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.--Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.     

A simplified procedure for preparation of undecalcified human bone sections

A new type of apparatus for sectioning samples of hard, undecalcified bone is described. Slices of fresh or archeological human bone 4-5 mm thick are dehydrated and then embedded in epoxy resin. The apparatus used to prepare sections from the resulting blocks consists of a low-speed rim-type diamond cut-off wheel and a slowly advancing table carrying the specimen held in a rotating mount. Sections may be cut at a thickness of 80 micron +/- 1%. After cleaning in an ultrasonic bath, these can be mounted on slides for quantitative microscopic examination with transmitted light. Grinding and polishing are not necessary. The results obtained are illustrated.     

A New Method for Preparation of Undecalcified Bone Sections

A new method for preparation of sections of undecalcified bone is described. Samples of ovine bone were embedded in methylmethacrylate and thick-sectioned with a cutoff machine or commercial band saw. Composite slides were prepared by gluing white acrylic to glass using cyanoacrylate glue. Bone sections were glued to the composite slide and then surface polished by grinding or ultramilling. The polished surface of the section was then etched and stained. The techniques described in this paper reduce the time spent grinding or milling sections and improve resolution of surface-stained features of undecalcified bone sections.     

A new method for preparation of undecalcified bone sections

A new method for preparation of sections of undecalcified bone is described. Samples of ovine bone were embedded in methylmethacrylate and thick-sectioned with a cutoff machine or commercial band saw. Composite slides were prepared by gluing white acrylic to glass using cyanoacrylate glue. Bone sections were glued to the composite slide and then surface polished by grinding or ultramilling. The polished surface of the section was then etched and stained. The techniques described in this paper reduce the time spent grinding or milling sections and improve resolution of surface-stained features of undecalcified bone sections.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

Summary We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([³H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [³H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [³H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.