A Rapid Method for Preparing Paraffin Serial Sections of Teeth and Their Supporting Structures in the Dog

A method of preparing dogs' teeth for serial sectioning involving the following steps is described. The tissues are fixed in 10% formalin followed by partial dehydration in 80% iso-propyl alcohol; decalcified in 5% nitric acid in 10% formalin; washed for 5-8 hours in running water and neutralized in 5% aqueous sodium sulfate for 24 hours, followed by further washing in running water for another 24 hours. They are then dehydrated over a period of 4 days in increasing grades of iso-propyl alcohol and embedded in tissue-mat in sub-atmospheric pressure of 10 lb. Sections so obtained were better suited for cytological study than celloidin preparations, and serial sections could be obtained in a much shorter time.     

Preparing Serial Sections of Mature Corn and Wheat Kernels

Methods are described for preparing serial sections of paraffin-embedded mature corn and wheat kernels. Prior to embedding corn kernels are killed and fixed in formalin-aceto-alcohol (FAA), then steeped 5 days in 50% glycerol. After embedding by a special procedure, a thin slice is cut from one side of the kernel and the first few cell layers removed. The exposed surface is submerged in 20% glacial acetic acid in 60% ethanol for 2 or 8 days depending on the surface exposed, 2 days in air at 100% relative humidity at room temperature, and 2 days in air at 100% relative humidity at 8°C, successively. Wheat kernels, fixed in formalin-aceto-alcohol and embedded by the regular paraffin procedure, are similarly trimmed to expose a surface which is submerged in 20% glacial acetic acid in 60% ethanol for 2 days, 2 days in air at 100% relative humidity at room temperature and 2 days in air at 100% relative humidity at 8°C, successively. The corn and wheat kernels prepared by these methods give good serial sections when cut as thin as 14μ. The application of these methods to other seeds and caryopses is suggested.     

Producing Frozen Sections of Calcified Bone

We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision. tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcel-lulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocom-pound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the “sectioning window” is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly “papered” with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide: adhesion is aided by “press-blotting” with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.     

Stained Ground Sections of Teeth and Bone

Formalin-fixed rat hemimandibles were ground to approximately 80μ with the aid of a diamond disc attached to a dental handpiece. A 1/10-hp motor attached to the handpiece by a pulley powered the disc to speeds up to 4000 rev/min. Contact between the hemimandible, which was placed on water-moistened cork, and the diamond disc was made possible through manipulation of the handpiece. The ground sections obtained by this procedure were stained with hematoxylin and eosin and periodic acid-Schiff for microscopic study. The fine structure of mineralized tissues (teeth and bones) as well as related soft structures, e.g., blood vessels, endosteum, pulp, etc., were demonstrated. The procedure did not require the embedding of the tissue nor were complicated grinding devices necessary.     

Organic Chelating Agents for Decalcification of Bones and Teeth

Lower jaws of adult guinea pigs were fixed in 10% neutral formalin 24 hours following intra-arterial and intra-man-dibular injection of the same fluid. They were then decalcified 3 weeks by 3 changes of Versene or Sequestrene (ethylene diamine tetracetic acid, disodium salt). Examination of the sections revealed complete decalcification, excellent fixation and selective staining of bone, cartilage, tooth and other tissues. Comparison with acid-decalcified preparations leads us to conclude that chelate-decalcification is at least as good if not superior to acid-decalcification in the preparation of such hard tissues.     

The Identification of Proteoglycans and Glycosaminoglycans in Archaeological Human Bones and Teeth

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.     

The Study of Barium Concentration in Deciduous Teeth,Impacted Teeth,and Facial Bones of Polish Residents

The study determines the concentration of Ba in mineralized tissues of deciduous teeth, permanent impacted teeth, and facial bones. The study covers the population of children and adults (aged 6–78) living in an industrial area of Poland. Teeth were analyzed in whole, with no division into dentine and enamel. Facial bones and teeth were subjected to the following preparation: washing, drying, grinding in a porcelain mortar, sample weighing (about 0.2 g), and microwave mineralization with spectrally pure nitric acid. The aim of the study was to determinate the concentration of Ba in deciduous teeth, impacted permanent teeth, and facial bones. The concentration of barium in samples was determined over the ICP OES method. The Ba concentration in the tested bone tissues amounted to 2.2-15.5 μg/g (6.6 μg/g?±?3.9). The highest concentration of Ba was present in deciduous teeth (10.5 μg/g), followed by facial bones (5.2 μg/g), and impacted teeth (4.3 μg/g) (ANOVA Kruskal-Wallis rank test, p?=?0.0002). In bone tissue and impacted teeth, Ba concentration increased with age. In deciduous teeth, the level of Ba decreased with children’s age.     

Three-Dimensional Reconstruction of a Dictyosome using Serial Sections

A single dictyosome from a suspension cultured sycamore cellhas been reconstructed on an enlarged scale using electron micrographsobtained from a series of 12 thin sections as the basis forthe moulding of profile slices. Slices were hardened and stucktogether to form single cisternae, tubules and vesicles. Thesestructural elements were joined, true-to-scale, to give a three-dimensionalfigure. The model thus obtained is more realistic than otherwell-known dictyosomal models previously presented in the literature.In contrast to these our model exhibits occasional membranecontinuities between adjacent cisternae of the dictyosomal stackand entertains the possibility of similar connections betweencisternae of the endoplasmic reticulum and the Golgi apparatus. Key words: Acer pseudoplatanus, Dictyosome reconstruction     

Preparing Epoxy Sections of Insect Tissues for Light Microscopy

Sections of aldehyde-fixed and osmium-stained insect tissues embedded in various epoxy resins were affixed to glass slides by use of a slide cover and hotplate combination. A high concentration of solvent vapor over the sections was thus maintained while they dried down on the slides, resulting in excellent flatness and adhesion. Sections were then stained at an elevated temperature with a mixture of equal parts of 3 dye solutions: 1% toluidine blue O, 1% safranin O, and saturated auramine O, all made up in 1% solution of borax in water. The method resulted in excellent differentiation of all insect tissue components including lightly chitinized structures.     

Modification of the Carmen-Faull-Pullar Racks for Free-Floating Serial Sections

The original staining racks designed by Carmen et al. (Stain Techn., 43: 157-60) have been redesigned to use inside plates of 0.06 inch thickness and outside (top and bottom) ones of 3/16 inch. The greater thicknesses permit freer circulation about the sections and avoid the need for outer clamps to hold the assembly together. As in the original racks acrylic sheeting has been used, but with 25 × 45 mm holes for sections in each plate. Ordinary fiberglass window screening was cemented to one side of each plate. The assembly of 12 inner and the 2 outer plates was held together by 2 bolts made of 1/4 inch acrylic rod. Since clamps on the edges of the assembly were not needed, smaller staining dishes could be used, with coincident economy in volume of staining solutions.     

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.     

The Rapid Preparation of Celloidin Serial Sections Following India Ink Injections

For the study of capillary penetration in the central nervous system of the chick embryo, following India ink injections, celloidin serial sections are superior to those prepared by the paraffin technic. The celloidin sections are arranged on a moist cigarette paper mat, which when filled is inverted and applied to a microscope slide so that the sections contact the glass surface. Subsequent to dehydration and clearing the sections are isolated on the slide by peeling off the cigarette paper. Forty-five minutes are required to prepare a slide of thirty sections from the time the block is trimmed until the cover slip is mounted with Clarite.     

An Abrasive Paper Technique for Preparing Sections of Wood for Microscopic Study

Dry wood specimens are sawed to 2mm thickness and impregnated with resin such as Lewisol 28 (Hercules Powder Co.). One side of the specimen is ground by hand on abrasive papers of grades #100, #180, #240, and #320. This side is then cemented to a petrographic glass slide with stick shellac and the other side similarly ground. Scratches can be eliminated by scraping the ground surface with the sharp edge of an ordinary glass microscopic slide. The section is removed by heating the slide, dissolving the shellac with alcohol and the resin of the embedding matrix with xylene. The sections can be stained in a hot saturated alcoholic solution of safranin O, counterstained with 0.01% fast green in an equal parts mixture of clove oil, methyl cellosolve, and absolute alcohol, and mounted in balsam.     

Floating-Out Technics for Rapid Placement of Ribbons of Serial Sections on Slides

The floating-out technic, popular for single paraffin sections, can be applied successfully to ribbons of serials by either of two procedures. (1) If spreading time for the sections is uncritical suitable lengths of ribbon for attachment to a slide are laid on water at a temperature about 8° C below the melting point of the paraffin and manipulated with a rubber bulb pipette to form a unit. This ensemble can then be picked up by the slide in much the same manner as a single section. (2) If spreading time is critical, as for objects that have had guide limes embedded with them, several ribbons are arranged on a cold, dry slide and transferred to the water as a unit. Placing the ribbons on the cold slide so that they slightly overhang one end and the sides of the slide allows them to make proper contact with the water as the slide is immersed. To facilitate controllable spreading in both methods, the water should have added to it 0.5 ml of albumen-glycerol adhesive per 100 ml. Adding water to the slide after the sections have been picked up or manipulation of the ribbons is generally unnecessary if the ribbons have been aligned accurately on the floating-out bath.