Rapid Three-Dimensional Reconstruction at the Light Microscopic Level and a Technique for Re-Embedding the Same Semithin Sections for Electron Microscopic Examination

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 μm sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.     

Demonstration of Sensory-Motor Innervation of Postmetamorphic Forelimb Regenerates of Triturus Alpestris (Urodela) on Cryotome Serial Sections Using Horseradish Peroxidase

A method has been developed to obtain horseradish peroxidase-treated serial sections containing spinal cord as well as bilateral ventral and dorsal roots, dorsal root ganglia and spinal nerves. Young postmetamorphic newts (Triturus alpestris) served as experimental animals. After cryotome cross sectioning the forelimb region of the trunk, slices 80 μm in thickness were mounted serially with up to 15 sections per slide. This facilitated subsequent staining manipulations and made partial loss of sections less likely.     

Sciatic nerve transection in neonatal rats induces apoptotic neuronal death in L5 dorsal root ganglion

Transection of a peripheral nerve in neonatal rats induces death of the axotomized neurons which may be due to either necrosis or apoptosis. In the present investigation, neuronal cell death in L5 dorsal root ganglion was evaluated after unilateral sciatic nerve transection in rats at 1, 3, 5, 7 and 10 days age. After 5 days, right (experimental) and left (control) dorsal root ganglia in all groups were removed, fixed, processed and embedded for either light or electron microscopy. Normal nucleoli were counted in paraffin embedded serial sections, and correction factors for split and multiple nucleoli were applied as well as the physical disector. The number of neurons in the right dorsal root ganglia, as compared with the controls, was significantly lower in all groups, and the percentage of the reduction at 1, 3, 5, 7 and 10 days was 32.4, 27.2, 23.8, 22.8 and 21.8% respectively. On the other hand, the results of neuronal counts using the disector method showed 34.0, 25.7, 20.2, 20.0 and 14.2% reduction in the number of neurons at 1, 3, 5, 7 and 10 days, respectively. The microscopic and ultrastructural results indicated that there were typical morphological changes similar to those of apoptosis, including condensed basophilic nuclei, formation of nuclear caps, cell shrinkage and apoptotic body formation. We concluded that there is an increase in apoptosis in dorsal root ganglia following sciatic nerve axotomy with the greatest neuronal loss on postnatal day 1.     

Axotomy-induced apoptosis in adult rat primary sensory neurons

Neuronal death following unilateral axotomy of a sensory nerve has long been inferred from neuronal counts of dorsal root ganglion neurons, using the contralateral ganglia as a control. The counting methods used usually involved the counting of neuronal nucleoli and made assumptions about them which could conceivably be flawed. Very few studies have used direct observations of dying or degenerating neurons to address questions concerning the duration of the period of neuronal death or the mechanisms involved in this process. Here we describe a morphological, morphometric and histochemical study into the nature and duration of sensory neuron death following transection and ligation of the sciatic nerve at mid-thigh level in the adult rat. We show that at least some of this neuronal loss occurs by apoptosis as defined by morphological criteria and in situ end-labelling of damaged DNA. Absolute numbers of apoptotic neurons were counted from serial paraffin sections of ganglia and estimates of neuronal numbers obtained by disector analysis at 1, 2, 3 and 6 months after axotomy. Using this approach we show that axotomy-induced apoptosis begins at around 1 week and continues up to at least 6 months after axotomy.     

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.     

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.     

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.     

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.     

Section Thickness and Grain Count Variation in Tritium Autoradiography

H² styrene-glycolmethacrylate is a suitable standard source for autoradiographic model studies. When it is used for quantitative autoradiography, the coefficient of variation for grain counts is minimal at a section thickness of 2.5 μ and greater. Variation increases with progressively thinner sections. When sections of the same thickness are mounted on separate slides, there is no significant variation in grain counts at the 95% confidence level when the slides are processed simultaneously.     

Improved Polyester Wax Embedding for Histology

Polyester waxes are fatty add esters of polyethylene glycol. Polyethylene glycol 400 distearate melts at 35°C, infiltrates tissues well, and sections readily at 2 μ to more than 30 μ. Sections 2 μ to 6 μ are more easily cut when a kitchen strainer full of solid CO₂ (dry ice) is mounted above the microtome to cool the block and the knife, and when the knife crosses the block very slowly. Ribbons are flattened in water at room temperature and are mounted conventionally. Polyester ribbons are somewhat stickier than paraffin ribbons. Polyethylene glycol 400 distearate is slightly hydrophilic; immediately after microtomy and before the ribbon is affixed to the microscope slide, sections in the wax ribbon may conveniently be stained with 0.05% toluidine blue in aqueous benzoate buffer, pH 4.4. Tissue structure is better preserved in polyester than in paraffin wax, probably because structural lipids are better retained and localized. However, this difference between waxes is slight if tissues are well fixed and dehydrated. Other advantages of polyester wax are that sections fragment less, hard tissues rarely split away from the wax ribbon, no static electricity is generated, and the microtome knife seems to remain sharp for a longer time.     

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.     

Single-section counting error when distinguishing between primordial and early primary follicles in sections of rat ovary of different thickness

The number of primordial follicles within an ovary is frequently determined by counting 5, 7 or 10 microns thick sections and multiplying by the fraction of sections counted and a correction factor to adjust for duplicate counts. The objectives of the present study were: (i) to evaluate the accuracy of the correction factor developed by Abercrombie (1946); (ii) to evaluate the accuracy of the classification of primordial follicles from single tissue sections; and (iii) to determine the incorporation rate of 5-bromo-2-deoxyuridine into primordial follicles. In Expt 1, rat ovaries were sectioned at a thickness of 5, 7 or 10 microns. Primordial follicles were counted and classified across ten adjacent ovarian sections. The percentage of primordial follicles from single sections that were counted twice was 10, 9 and 2% in 5, 7 and 10 microns sections, respectively. This was lower than predicted by Abercrombie's method. The major error in counting from single sections was classification of early primary follicles as primordial follicles (55, 33 and 3% in 5, 7 and 10 microns sections, respectively). In Expt 2, a mean of 12 +/- 7% of primordial follicles incorporated 5-bromo-2-deoxyuridine after infusion for 7 days (four of seven rats had no labelled primordial follicles). In conclusion: (i) Abercrombie's correction factor should not be used for adjusting counts of follicles; (ii) evaluation of primordial follicles from single sections gives inaccurate counts and incorrect classification is of greater importance than duplicate counting, particularly in thinner sections; (iii) for evaluation of the number of follicles, 10 microns is the optimal thickness; and (iv) primordial follicles incorporated 5-bromo-2-deoxyuridine infrequently.     

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.     

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.     

Improved Paraffin Sectioning with Etched Microtome Knife Edges

Carbon steel microtome knives etched with 0.1 N HNO₃ for 2 min display a very sharp cutting edge. Over a period of 3 yr no damage to the steel has been detected. The effect on paraffin sectioning was observed by comparing acid-treated knives with nonetched but well-sharpened ones. Sections of whole eyes cut with an etched blade showed approximately 15% less compression of the parffin matrix than those sectioned with an untreated knife. Tissues selected from routine autopsy material presented approximately 9% reduction in compression. As a result, excellent ribbons of sections could be cut from 5-7 μ and floated on water at 42—46 C with a minimum of folds or distortion. Etching improved sectioning when knife edges having bevel angles in a range of 31-39° were used, and also when the bevel was decreased to 20°, but the 20° edge gave impractically short service.     

Contributions to Semithin Sextioning on A Conventional Rotary Microtome

Procedures for obtaining sections 1 μ thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen bolder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections I micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

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.     

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.     

Comparing the somal size and nuclear positions of the monkey stellate and coeliac ganglion cells

The stellate and coeliac ganglia of 2 Macaque monkeys were cut serially at 1 micron thickness and analysed. Results from the analysis of 82 stellate and 60 coeliac ganglion cells in 1 monkey show that in cross-sections, the neuronal nuclei may be eccentric, centric or nearly centric and remain so throughout the longitudinal extent of the neuron. In both ganglia, the majority of neurons possess eccentric nuclei, but in the coeliac ganglion, the percentage of neurons with centric and/or nearly centric nuclei is higher (41.7%) than that in the stellate ganglion (26.3%). While 5% of neurons in the coeliac ganglion are binucleated, no binucleated neurons were found in the stellate ganglion. The somal size ranges of the stellate (10-39 microns) and the coeliac (14.5-45 microns) ganglion neurons as obtained from both monkeys are quite close. The percentage frequency distribution of the stellate ganglion neurons in monkey 1 was also quite similar to that of the coeliac ganglion neurons. It is concluded that different neuronal size is not likely to be associated with different target organs.