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.     

In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys

Recent evidence suggests that a local reninangiotensin system is operational in the kidney and that it mediates some of the actions of angiotensin II on renal tubules. In this study the ontogeny and renal distribution of the unique precursor to angiotensin II formation, angiotensinogen, was investigated in rats by use of immunohistochemistry, immuno-electron microscopy and non-isotopic hybridization histochemistry. At the light-microscopic level, intense staining for angiotensinogen was found in the proximal convoluted tubules of the cortex, with lighter staining in the straight proximal tubules of the outer stripe. The strongest immunostaining was found in the kidneys of neonatal rats, where glomerular mesangial cells and medullary vascular bundles were also immunopositive. The angiotensinogen content of the kidneys in late gestation embryos and neonates showed the presence of angiotensinogen by day E18 and a peak content in the neonate. Non-isotopic hybridization histochemistry with biotinylated oligodeoxynucleotide probes confirmed the presence of angiotensinogen mRNA expression in the proximal convoluted tubules of the renal cortex. Electron-microscopic immunohisto-chemistry showed staining of relatively few electron-dense structures close to the apical membrane of proximal convoluted tubule cells in the adult kidney. In the neonatal rat kidney, angiotensinogen immunostaining at the electron-microscopic level was found throughout the proximal tubule cells and was markedly stronger than that seen in adult kidney. The presence of angiotensinogen, from embryonic day 18, in the proximal tubules, mesangial cells and vasculature of the kidney suggests multiple potential sites of intrarenal angiotensin II generation with an ontogeny in late gestation.     

Histological analysis of undecalcified thin sections of archeological bone

Archeological bone often lends itself to histological analysis. Sections from bone samples approximately one thousand years old may show as much structural preservation as those only a few hundred years old; hence, it appears that the degree of preservation is not necessarily affected by time. Enough structure may be preserved to permit the diagnosis of metabolic disorders of bone which might go undetected by other methods. This type of analysis can be utilized to accept or reject individual remains suspected of being pathological on the basis of other, less precise diagnostic techniques.     

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.     

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.     

Surface staining of sawed sections of undecalcified bone containing alloplastic implants.

A simple new method is described for the histological evaluation of bones containing alloplastic implants of ceramic and/or metallic materials. The undecalcified bone is embedded in acrylic resin and sectioned at 50-200 micrometer using a sawing microtome. One surface of the preparation is stained up to 10 micrometer deep by floating the preparation on Giemsa stain. Other staining procedures are possible. Microscopic detail is satisfactory for histological and morphometric evaluation.     

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.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

Summary We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electronmicroscopical level.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding reserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electron-microscopical level.     

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.     

In situ 3D visualization of biomineralization matrix proteins

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.     

In situ interaction of cartilage proteoglycans with matrix proteins

The interaction of proteoglycans with other matrix proteins via thiol-disulphide interchange was explored. Chick sternal cartilage was extracted with 4 M guanidine hydrochloride in the presence and absence of N-ethylmaleimide and the proteoglycans from the centrifugation A2 fractions were isolated. Those from extracts without N-ethylmaleimide were linked with reducible bonds with 10-15 proteins-glycoproteins including the link proteins, the 148 kDa and 36 kDa proteins. The same was observed with extracts of pig laryngeal and sheep nasal cartilage. The linked proteoglycans from sheep amounted to 2-3% of the extractable uronic acid and belonged to two populations. The major fraction was included by Sepharose 6B (Mr 110 000) had twice as long chondroitin sulphate chains, higher 4-sulphated residues and a high content of aspartic acid and leucine-rich protein. The larger proteoglycans had a size and composition similar to those of aggregating proteoglycans.     

An improved method for preparing cryostat sections of undecalcified bone for multiple uses.

We developed a technique that permits the use of serial sections (7-20 microns) from a single fixed piece of bone tissue for immunofluorescence, measurement of fluorescent bone labels, enzyme histochemistry, and general staining. This technique combines modifications of previously established methods with perfusion of the polymer polyvinylpyrrollidone (PVP) to improve sectioning, and produces reliable sections with good preservation of both hard and soft tissues. The combination of techniques from several workers, the use of perfusion with a polymer to increase the sectionability of the bone, and the addition of a gelatin adhesive on top of pressure-sensitive adhesives represent a significant improvement over previously described methods. The sections obtained are usable for immunocytochemistry, general staining, enzyme histochemistry, and visualization of fluorescent bone labels. We have consistently used tissues prepared in this manner for immunohistochemical demonstration of neuropeptides in skeletal tissues and for localizing tartrate-resistant acid phosphatase (TRAP). In addition, other tissues obtained from PVP-perfused rats, such as brain, spinal cord, muscle, gut, and sympathetic ganglia, are also well preserved and demonstrate immunohistochemical staining comparable to and possibly superior to that obtained with normal fixation protocols.     

In situ hybridization: use of 35S-labeled probes on paraffin tissue sections

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.