Improvements in histological techniques for epoxy-resin embedded bone specimens

A procedure is presented in which some of the processing difficulties with fixation, embedding and cutting whole mouse bones and large bone pieces from other species are considered. The bone specimens are fixed in acetone or by a Karnovsky-formol-saline process which preserves intact endosteal surface-to-cortex layers. After fixation the bones are embedded in a hard mixture of epoxy resin to provide blocks with face sizes up to 3.5 x 3.0 cm. Mineralized sections are cut to 4 micrometer; demineralized at 3 micrometer. Sections are fastened to gelatin-subbed slides with pressure plates which produce flat, secure sections. After removal of the plastic, an unmodified Mayer's hematoxylin and a polychromatic eosin staining method is applied to demineralized sections, and a slightly modified method to mineralized sections.     

A Method for the Preparation of Undecalcified Bone Sections for Light Microscopy and Microradiography

A method which gives good quality 1-2 μm thick sections of undecaldfied cancellous and thin cortical bones for light miuoscopy is described. Formalin fixed material is dehydrated in graded acetones and embedded in a modiEed formula of Spurr's low viscosity embedding medium. After a 16 hour polymerisation period at 60 C, sections are cut at 1-2 μm thickness on a Porter-Blum JB4A rotary microtome Using glass knives. Sections are attached to clean glass slides with heat, the resin degraded in bromine vapour and removed in acetone. This allows comparative ease of staining. The technique is rapid, does not interfere with tetracycline fluorescence and the same specimens can be used to prepare thick sections for microradiography.     

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 section at 50-200 μm using a sawing microtome. One surface of the preparation is stained up to 10 μm deep by floating the preparation on Giemsa stain. Other staining procedures are possible. Microscopic detail is Satisfactory for histological and morphometric evaluation.     

A Demineralization Procedure for Enzymatic Histochemical Use: A Quantitative Succinic Dehydrogenase Assay

Thinnest practical slices of bones or teeth are suspended at 4 C in a 10% solution of EDTA in 0.1 M tris buffer brought to pH 6.95 with KOH pellets. The solution is stirred moderately and continuously with a magnetic stirrer until specimens are demineralized. Unwashed specimens, taken directly from the demineralizing fluid, are frozen on a block of CO₂ ice, mounted on a tissue carrier and sectioned at 6 μ in a cryostat. Histochemical stains conducted on sections stored in a slide holder enclosed in a plastic bag in a refrigerator for as long as 2 wk were successful. Quantitative studies on the preservation of succinic dehydrogenase showed nearly all of it present in specimens demineralized for 2 days, and approximately 50% remaining in specimens demineralized for 7 days. Qualitative studies with other dehydrogenases indicate that several may be similarly affected.     

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.     

Paraffin Section Thickness—A Direct Method of Measurement

Paraffin section thickness may be directly measured by re-embedding the sections wider consideration, cutting them again at right angles to the original plane of sectioning, and taking direct measurements with a filar micrometer after staining and mounting. Conditions and materials with which relatively un-distorted 3 and 5 μ sections were secured include (a) a hand-honed knife with a 23° facet bevel, set at a clearance angle of 7°, and (b) a hard paraffin (56-58°) embedding medium, preferably with 5% beeswax and 5% bayberry wax added. By taking direct measurements, it was found that bull testis tissue cut at a microtome setting of 10μ averaged 10.82 μ in thickness. Settings of 5 μ and 3 m resulted in sections averaging 5.25 and 3.31 μ in thickness respectively. Stages in sporogenesis of Onoclea sensibilis, Lewitsky fixed, were examined after sectioning at settings of 10, 5, and 3 μ to determine necessity for thin sections. For this material, it was indicated that mitochondrial preparations as thick as 10 μ were worthless, regardless of good fixation and proper staining. Three-micron sections give the best results.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

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.     

Identification of Normal or Neoplastic Murine Mesenchymal Cells in Chimeric Tissues or Heterotransplants

An improved method for identifying murine mesenchymal cells in chimeric tissues or heterotransplants using Hoechst dye 33258 is described. Following fixation in formalin-saline, tissues are embedded in JB-4 plastic Sections 3 μm thick are then stained in a 10 μg/ml solution of Hoechst 33258 in Hanks' balanced salt solution for 5-10 min at 4 C. After rinsing, the sections are coverslipped using a modified polyvinyl alcohol mounting medium. This approach offers several advantages over existing techniques: 1) uniform section thickness is more easily obtained than with paraffin or cryostat microtomy, thereby allowing improved resolution and more reliable identification of mesenchymal cells with small nuclei such as skeletal muscle myocytes or fibroblasts, 2) the preparations are stable over long periods and can be repeatedly viewed or photographed, and 3) calcified tissues can be examined without prior decalcification. An example is shown of species identification using rat chondrosarcoma cells grown in nude mice.     

Rapid Nissl Staining for Frozen Sections of Fresh Brain

Working with X-ray film autoradiography of soluble isotopes, we needed a staining technique for the localization of nuclei in frozen sections of fresh brain. We have found no Nissl staining method in the literature concerning autoradiography specially recommended for this purpose, nor have we found in handbooks on staining a Nissl method clearly recommended for unfixed, frozen sections of brain. The methods described are intended for paraffin or celloidin sections, and require fixation of brain before sectioning (which must be avoided when working with soluble isotopes). Because autoradiography is a time-consuming method, any technique which shortens time needed for the overall procedure is welcome. Most Nissl techniques described in the literature require long preprocessing of the tissue. We found two rapid methods, described by Humason (1967) and LaBossiere and Glickstein (1976), but their application to frozen sections did not give good results. After trials with several types of techniques, we succeeded in developing two Nissl modifications with slightly different qualities, one of 12 min and the other of 2-3 h. The longer method includes conventional steps in staining; the shorter method does not include fixation or lipid extraction. These methods were applied to 20-60 μm brain sections cut in the cryostat at -10 to -12 C and dried on gelatinized slides.     

Enzyme, Lectin and Immunohistochemistry of Plastic Embedded Undecalcified Bone and other Hard Tissues for Light Microscopic Investigations

Enzymes and tissue antigens were localized on plastic embedded undecalcified bones and teeth using Technovit 7200 VLC (Kulzer, Germany). This resin is hard enough for cutting and grinding procedures on rotating plates with diamond layers. The pores between the diamond grains are not obstructed with this resin. The procedure described here permits localization of antigens in the soft tissues adjacent to, or in the biological hard tissues themselves and in dental implants (ceramic or metallic) on the light microscopic level. The undecalcified bone is fixed and embedded in plastic and cut at 100-150 μm. The slices are ground automatically by a grinding machine to a thickness of 5-10 μm. After application of the substrates for alkaline and acid phosphatases and the required dyes, the distribution of these enzymes can be demonstrated. Tissue antigens also can be detected with slightly modified standard techniques of immunohistochemistry and lectin histochemistry using the peroxidase technique or fluorescence microscopy.     

A Method to Stain Decalcified Bone without Loss of Structural Detail

A modification of Bodian's protargol-S technique is done on 7 μm sections of decalcified bone. Light microscopic results are greatly improved when compared to either ground bone sections or decalcified bone stained routinely with hematoxylin and eosin.     

Finite element analysis of trabecular bone structure: a comparison of image-based meshing techniques

In this study, we investigate if finite element (FE) analyses of human trabecular bone architecture based on 168 μm images can provide relevant information about the bone mechanical characteristics. Three human trabecular bone samples, one taken from the femoral head, one from the iliac crest, and one from the lumbar spine, were imaged with micro-computed tomography (micro-CT) using a 28 μm resolution. After reconstruction the resolution was coarsened to 168 μm. First, all reconstructions were thresholded and directly converted to FE-models built of hexahedral elements. For the coarser resolutions of two samples, this resulted in a loss of trabecular connections and a subsequent loss of stiffness. To reduce this effect, a tetrahedral element meshing based on the marching cubes algorithm, as well as a modified hexahedron meshing, which thresholds the image such that load carrying bone mass is preserved, were employed. For each sample elastic moduli and tissue Von Mises stresses of the three different 168 μm models were compared to those from the hexahedron 28 μm model. For one sample the hexahedron meshing at 168 μm produced excellent results. For the other two samples the results obtained from the hexahedral models at 168 μm resolution were poor. Considerably better results were attained for these samples when using the mass-compensated or tetrahedron meshing techniques. We conclude that the accuracy of the FE-models at 168 μm strongly depends on the bone morphology, in particular its trabecular thickness. A substantial loss of trabecular connections during the hexahedron meshing process indicates that poor FE results will be obtained. In this case the tetrahedron or mass-compensated hexahedron meshing techniques can reduce the loss of connections and produce better results than the plain hexahedron meshing techniques.     

A Simple Technique for Osmicating and Flat Embedding Large Tissue Sections for Light and Electron Microscopy

Many investigators now use thin hand-sliced, tissue chopper, or Vibratome sections of fresh tissue in various procedures. In our experience brain and nerve sections varying in thickness from less than 40 to more than 300 μm, with or without prior embedding in agar, have a tendency to roll up or curl during aldehyde fixation and buffer washes. Once osmicated, such curled sections cannot be flattened. When the entire cut face of such thin slices is to be studied, sufficiently flat embedding so that some regions are not completely sectioned before others are even sampled is critical. This report describes fixation and flat embedding procedures, developed for light and electron microscopic autoradiographic studies of plastic embedded brain slices about 200 μm thick (Schwartz 1981), which can be applied to any comparable thin slice of nervous tissue (or potentially of many other tissues) to achieve maximally flat tissue faces. Since osmicated tissue slices are usually too thick to be transilluminated for direct examination with the light microscope, the methods described simplify preparation of the semithin sections required for this purpose.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Staining and Histomorphometry of Microcracks in the Human Femoral Head

We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 μm) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 μm) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways.     

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.     

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 μm ± 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.     

Thin Histological Sections Prepared from Large Thick Sections: a New Technique

A method is described for obtaining thin (1 μm) sections for light microscopy from large area thick (100 μm) sections of low viscosity nitrocellulose embedded specimens of human spinal osteoligamentous material.     

Hematoxylin Toluidine Blue-Phloxinate Staining of Glycol Methacrylate Sections of Retina and Other Tissues

For a detailed study of the developing chick retina a technique has been developed using glycol methacrylate embedding and a hematoxylin toluidine blue-phloxinate stain. After removal of the vitreous body, one half-segment of the eye is dehydrated through graded ethyl alcohols to 95%, infiltrated and embedded in glycol methacrylate, and sectioned at 2 μm. The sections are stained in alum hematoxylin and then in a mixture containing toluidine blue-phloxinate from a stock solution of the dye that has matured for 2-3 weeks. Differentiation is not required and there is only slight staining of the plastic matrix. The quality and clarity of the sections contrasts markedly with that of similarly stained 5 μm paraffin wax sections of the retina. This technique has also been applied to skin, spinal cord, dorsal root ganglia, pancreas and small intestine. The stained sections from these tissues have proved very useful in revealing structural components.