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.     

Embedding in Epoxy Resins for Ultrathin Sectioning in Electron Microscopy

Fixed tissue is dehydrated with tertiary butyl alcohol overnight. The following day it is cleared in toluene, infiltrated and embedded in Araldite resin-hardener-accelerator mixture without dibutyl phthalate, and polymerized at 60° C. More rapid than previous techniques, this method gives blocks which do not fracture unduly on trimming and provides sections of soft tissues at 1 μ for phase contrast microscopy, as well as ultrathin sections which cut as easily with glass knives as sections of methacrylate. Araldite manufactured in the U.S.A. and in England are different. Satisfactory proportions for the American are: hardener DDSA, 3.5 ml; casting resin 6005, 5.0 ml; accelerator B, 0.12 ml. For the British product, these are: hardener 964 B, 5.0 ml; casting resin M, 5.0 ml; accelerator 964 C, 0.25 ml. The use of 2% agar for orienting small specimens in Araldite is feasible. Mallory's borax-methylene blue has been applied to the staining of Araldite sections as thin as 0.5 μ mounted on glass slides.     

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.     

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.     

Histochemical identification of osteoclasts. Review of current methods and reappraisal of a simple procedure for routine diagnosis on undecalcified human iliac bone biopsies

Osteoclasts are known to have a high acid phosphatase content. We have adapted the simple simultaneous mono-coupling azo-dye method of Grogg and Pearse to undecalcified bone sections. A cold embedding in a mixture of glycol and methyl methacrylate was shown to well preserve the enzyme activity. Sodium alpha-naphtyl phosphate (1 mg/ml) and fast violet B (2 mg/ml) are used in 0.1 M acetate buffer, pH 5.0. The addition of 1 mM L(+) sodium tartrate selectively inhibits the acid phosphoprotein phosphatase ("osteoblastic acid phosphatase") but not osteoclastic lysosomal acid phosphatase. Counterstaining with phosphomolybdic aniline blue WS leads to well contrasted sections, providing accurate measurements of osteoclast number.     

Ageing domestic sheep (Ovis aries L.) from growth lines in the cementum of the first incisor

First incisor teeth from known‐age domestic sheep were examined to determine whether annual lines developed, and to test 3 processing techniques. Fluorescing lines in undecalcified, unstained transverse sections indicated completed years in 44 % of teeth. Dark lines in decalcified, stained transverse sections indicated completed years in 8 % of teeth. Dark lines seen by reflected light in undecalcified roots ground transversely by hand indicated completed years in 62 % of teeth. Grinding by hand was the simplest, quickest, and most dependable method.     

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.     

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.     

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.     

Staining protocols for human pancreatic islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.     

A simple method for preparing thin (10 microM) histological sections of undecalcified plastic embedded bone with implants

A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface.     

A Simple Method for Preparing Thin (10 μm) Histological Sections of Undecalcified Plastic Embedded Bone with Implants

A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface.     

Development of the nasal septum in rabbits. III. Quantitative observations on cells and intercellular substance

With the aim to further characterize the various regions of the nasal septum, the following measurements were made: In the thin anterior region and in the thick regions in the middle and posterior parts of the septum the quantitative relationships between cells and intercellular substance as well as the size of the cells were determined. The material used, included transverse and horizontal sections through the nasal septum of rabbits of ages of 1 day and 2, 4, 6, 8, 12 and 24 weeks. The results showed that in the thin anterior region the relationship between cells and intercellular substance remains constant, while in the two thick regions the amount of intercellular substance proportionately increases. In the thick regions, the cells are always larger than in the thin region. The rapid growth of the cells between the 4th and the 6th week coincides with a growth-spurt of the whole head.     

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 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.     

Immunohistochemical demonstration of various antigens in tissues embedded in plastic

In the course of a chronic experimental study of polyarthritis in rats, we have established a plastic embedding technique which allows the processing not only of soft tissues but also of tissues such as cartilage, intraarticular ligaments, and even undecalcified whole rat joints and provides broad survival of enzymes and antigens. Preservation of morphological details and the precision of antigen localization are superior to those seen in cryostat and paraffin sections. The method seems to have much diagnostic and investigative value, especially in arthritis research.     

Micro-biomechanics vs macro-biomechanics in cortical bone. A micromechanical investigation of femurs deformed by bending

Previous studies have shown that longitudinal and transverse lamellae in compact bone signal the presence of tensile and compressive forces, respectively. On this basis an investigation has been carried out to ascertain the distribution of lamellae in femoral shafts that have been deformed by bending. To do this, a series of undecalcified plane-parallel sections 100 microns thick were prepared, and the proportion of transversely oriented collagen and crystallites in the lamellae was measured, using circularly polarized light as an illuminating source and a Quantimet 720 image-analyzing computer. It has been concluded that the distribution of the two types of lamellae is in accordance with the need to compensate for the deformity produced by the bending of the bones, and that an obvious relationship exists between the macroscopic features of the femurs and their modified microscopic structures.     

Sectioning of Undecalcified Bone; with Special Reference to Radioautographic Applications

A rapid technic for the preparation of 6 μ serial sections of undecalcified bone is described. The specimen is fixed and dehydrated in acetone and ether. It is then treated with a 1:1 mixture of the monomers of ethyl and n-butyl methacrylate catalyzed with benzoyl peroxide. The monomers are removed with ether and the ether is removed under vacuum. Infiltration is accomplished under vacuum using a partial polymer of the same mixture of monomers. Polymerization is completed in 36 hours under pressure at 50°C. The tissue is sectioned with a heavy-duty microtome, the sections are mounted on glass slides and nuclear emulsions applied. Young and adult bone of laboratory animals and man have been cut successfully. Microscopic structural detail is preserved and there is no evidence of translocation of the radioactivity.     

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.