True serial-sectioning of fossil material

A method of cutting serial sections of fossil material using a very thin diamond annular saw, clamped under tension and revolving at 3000 rev/min is described. A small skull of the therapsid reptile Endothiodon has been cut into 104 serial sections 0–6 mm thick. The technique is still in its infancy, but is described to show its potentialities.     

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.     

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum.

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.     

The routine sectioning of undecalcified bone for cytochemical studies

Synopsis A method is described by which serial sections of fresh, undemineralized adult bone can be obtained. Such sections are suitable for cytochemical investigations and can expedite diagnostic histology.     

Computer-aided stereographic representation of an object reconstructed from micrographs of serial thin sections

A method is described which allows a three-dimensional object to be reconstructed from micrographs of serial thin sections using computer graphic techniques. The reconstructed object, which can be rotated three- dimensionally, is displayed on a colour visual display unit and the surface of the object is shaded in order that it can be observed to provide an illusion of a three-dimensional structure. Moreover, the technique makes it possible to represent an inner structure when seen through an outer one, also to observe other sectioned face views. The method as described here allows rapid visual evaluation of the results of three-dimensional reconstruction from serial thin sections when recorded with the aid of a light or an electron microscope.     

A Device for the Precise Transfer of Serial Sections for Electron Microscopy

A device based on a standard stereotaxic instrument, in which a Formvar-dipped loop carrying serial thin sections, and a rotatable grid holder are mounted in separate electrode holders, thus permitting accurate positioning of each with respect to the other and the precise transfer of the sections to the grid, is described and illustrated.     

ULTRASTRUCTURAL INTERRELATIONSHIPS OF NERVE PROCESSES AND SMOOTH MUSCLE CELLS IN THREE DIMENSIONS

The muscularis externa of the intestinal wall of frogs was fixed in osmium tetroxide, embedded in Vestopal-W, serially sectioned for electron microscopy, and stained with uranyl acetate. A method to obtain individually mounted and properly positioned serial sections is described. The three-dimensional techniques used during the course of this investigation demonstrate that it is possible to examine carefully relatively large areas of tissue on individual serial sections with the electron microscope and subsequently to construct montages of electron micrographs of pertinent areas from each section. Several carefully rendered interrelationships of nerve processes and smooth muscle cells in three dimensions are exhibited and described. Recent studies of other neuro-effector relationships are discussed in relation to the present status of the nature and organization of the autonomic nervous system in visceral organs.     

Optimization of dopaminergic neuron counting in substantia nigra of parkinsonian mice

Parkinson’s disease (PD) results from the degeneration of dopaminergic (DA-ergic) neurons of substantia nigra pars compacta (SNc). The disease is modeled in mice by the administration of a neurotoxin precursor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Neurotoxin efficiency is estimated by reduced number of DA-ergic neurons in SNc. Cell counting on serial sections is very laborious and expensive and, therefore, is not widely used in spite of its high precision. The well-known Konigsmark’s formula (KF) allows one to perform counting on sections chosen with a certain interval rather than to utilize serial sections. However, its precision decreases with increasing interval and other parameters. In this paper, we described the mathematical method of approximation (MA) by improving KF. MA maintains counting precision and allows one to reduce the time and expenses for material processing and analysis.     

The sessile drop method for immunohistochemical processing of unmounted sections of nervous tissue

A novel method for the immunohistochemical processing of free-floating tissue sections is described. Sections are immersed within drops of solution arranged on a hydrophobic surface. The procedure consists of sequentially suctioning away one fluid drop and replacing it by another, while the section remains in place. The technique permits easy testing of different antiserum dilutions, comparisons among different immunohistochemical protocols, and comparison of different antisera on serial tissue sections. Comparison is made to processing mounted sections.     

Interpretation of electron micrographs of single and serial sections

A method of securing serial sections for electron microscopy is described. Serial sections present certain anomalies of interpretation of a nature such that a complete and detailed three-dimensional reconstruction of the sectioned tissue cannot be made. These anomalies are discussed, as well as those which have been encountered in the interpretation of single sections. Observations of the following kinds have been made in an attempt to elucidate the interpretation of single and serial sections: differing methods of mounting adjacent sections, observation of the same section by high-angle stereoscopy, and examination of sections which have been shadowed prior to and subsequent to electron microscopy. It is found that the appearance of sections is independent of the choice of side to be placed against the formvar films. Stereoscopy shows that the appearance of fine structures is strongly dependent upon the direction of the penetrating electron beam with respect to the plane of the structures. Stereoscopy, combined with shadowing, shows quantitatively that extensive sublimation of polymer occurs upon normal exposure in the electron microscope. Observation of sections shadowed prior to electron microscopy indicates that varying amounts of material are removed between sections by the action of microtomy; i.e., it is probable that the sum of the thicknesses of several serial sections is considerably less than the total thickness of material removed from the block. It is believed that this effect, combined with the effect of sublimation, aids in explaining the failure of adjacent sections to exhibit continuity in their detailed structures.     

INTERPRETATIONS OF ELECTRON MICROGRAPHS OF SINGLE AND SERIAL SECTIONS

A method of securing serial sections for electron microscopy is described. Serial sections present certain anomalies of interpretation of a nature such that a complete and detailed three-dimensional reconstruction of the sectioned tissue cannot be made. These anomalies are discussed, as well as those which have been encountered in the interpretation of single sections. Observations of the following kinds have been made in an attempt to elucidate the interpretation of single and serial sections: differing methods of mounting adjacent sections, observation of the same section by high-angle stereoscopy, and examination of sections which have been shadowed prior to and subsequent to electron microscopy. It is found that the appearance of sections is independent of the choice of side to be placed against the formvar films. Stereoscopy shows that the appearance of fine structures is strongly dependent upon the direction of the penetrating electron beam with respect to the plane of the structures. Stereoscopy, combined with shadowing, shows quantitatively that extensive sublimation of polymer occurs upon normal exposure in the electron microscope. Observation of sections shadowed prior to electron microscopy indicates that varying amounts of material are removed between sections by the action of microtomy; i.e., it is probable that the sum of the thicknesses of several serial sections is considerably less than the total thickness of material removed from the block. It is believed that this effect, combined with the effect of sublimation, aids in explaining the failure of adjacent sections to exhibit continuity in their detailed structures.     

A simple method for collecting and mounting ribboned serial sections of epoxy embedded specimens

A simple and rapid method of handling ribboned serial sections of epoxy embedded specimens is described. Ribbons are cut from a block having the leading and trailing sides coated with contact cement. A scoop made from polyethylene tubing is used to remove a ribbon of sections from the boat of a glass or diamond knife and to transfer it to a pool of water on a microscope slide. Many ribbons (comprising hundreds of sections) can be mounted on a single slide. This method requires the construction of only one simple, inexpensive tool, the polyethylene scoop, and otherwise utilizes only items commonly available in the laboratory.     

A Simple Method for Collecting and Mounting Ribboned Serial Sections of Epoxy Embedded Specimens

A simple and rapid method of handling ribboned serial sections of epoxy embedded specimens is described. Ribbons are cut from a block having the leading and trailing sides coated with contact cement. A scoop made from polyethylene tubing is used to remove a ribbon of sections from the boat of a glass or diamond knife and to transfer it to a pool of water on a microscope slide. Many ribbons (comprising hundreds of sections) can be mounted on a single slide. This method requires the construction of only one simple, inexpensive tool, the polyethylene scoop, and otherwise utilizes only items commonly available in the laboratory.     

A morphological study of the anterior nervous system of the praesoma of Neoechinorhynchus cylindratus (Acanthocephala: Neoechinorhynchidae)

The nerve pathways in the praesoma, based on light microscopy of serial transverse, sagittal, and longitudinal sections stained with Ehrlich's acid hematoxylin are described for the first time for a memeber of Neoechinorhynchus. The route from the cerebral ganglion to the musculature and sense organs of the proboscis and body wall for 11 nerves, five pair and one single, the presence and structure of the Stutzzelle (support cell) and its association with the neck sense organs are described. A comparison with the nervous system in the praesoma of Paulisentis fractus is discussed.     

Method for Staining and Washing Thin Sections on Support Films

A procedure is described for staining large numbers of thin sections on support films for use with one-hole grids. The film is picked up, carried and protected using easily made plastic blocks. Loop-tipped forceps are then used to transfer tissue ribbons from the knife boat to the support film. A large number of tissue sections can then be stained and washed simultaneously in a modified Pyrex dish without damaging the film. After staining, the slot in the one-hole grid is centered over the tissue ribbon, and the grid is attached to the film. The method is suitable for serial reconstruction and the unobstructed viewing of large thin sections in the TEM.     

The direct measurement of cytochrome P450 in unfixed tissue sections

Summary A method for the measurement of cytochrome P450 in unfixed cryostat sections is described. The sections are incubated for 10 minutes at room temperature in a buffered solution containing polyvinyl alcohol and sodium dithionite. Two incubations are performed on serial sections, one in nitrogen and the other in carbon monoxide. Readings are taken on a Vickers M85 microdensitometer fitted with a high sensitivity photomultiplier amplifier system, the measurements being made on corresponding fields in the serial sections. Subtraction of the nitrogen values from the carbon monoxide values, after allowing for an absorption shift, gives the absolute spectrum of cytochrome P450. The subtraction corrects for the tissue content of other haem-containing proteins. The cytochrome P450 spectrum shows a sharp maximum at 450 nm, and two other minor components absorbing at 444 nm and 458 nm. The content of cytochrome P450 in animals fed with phenobarbitone was 2.4 times greater than in control animals.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

The estimation of the nucleic acids of tissue sections and its use as a unit of comparison for quantitative histochemistry

Summary There are many advantages in using the nucleic acid content of tissue sections as the unit of comparison for quantitative histochemistry. A quick, simple, reliable method for the estimation of DNA and RNA in sections of rat liver is described. The method depends on the differential hydrolysis in IN HCl at 60° C; the RNA is removed after 4 min hydrolysis and the DNA by a further 30 min hydrolysis. The optimum extinction of the hydrolysates is measured in an ultraviolet spectrophotometer. The results show a reproducibility, between serial sections, of better than ±5 %.     

A Plastic Embedding Technique for Analyzing Fluorescent Dextran-Amine Labelled Neuronal Profiles

A plastic embedding technique employing fluorescently labelled dextran-amines is described. After application of tracer to cut nerves and appropriate transport time, animals were fixed in paraformaldehyde. Subsequently their brains were dissected, heads and brains were dehydrated, embedded in methacrylate and sectioned serially on a rotary microtome. Plastic sections allow high resolution of single neuron profiles and complete serial reconstruction of un-distorted sections, including embryos with large amounts of yolk. In conjunction with whole mount analysis and double labelling, this technique can accurately reveal the spatial relationships of nerve components throughout development.