Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea

We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 microns-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA content of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry fo individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA content of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.     

Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea

Summary We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 m-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA contnet of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry of individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA contnet of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.     

Electron microscope autoradiography of DNA synthesis in the replication band of two hypotrichous ciliates

The synthesis of DNA in two hypotrichous ciliates, Styx sp. and an amicronucleated strain of Oxytricha sp., was studied by high voltage (1000 kV) electron microscopy. High voltage EM permits use of thick sections (0.25-0.40 micron), including serial sections; thick sections produce strong autoradiographic images with relatively short exposure times. The autoradiographs show that DNA synthesis occurs in a narrow part of the rear zone of a replication band in the macronucleus. Macronuclear DNA synthesis occupies a substantial part of the interdivision interval, and micronuclear DNA synthesis in Styx sp. takes place in early prophase at a time when macronuclear DNA synthesis is in its terminal phase.     

W10BSmL, a mutant of Euglena gracilis var. bacillaris lacking plastids

Organized proplastid structures are absent from dark-grown and light-grown cells of Euglena gracilis Klebs var. bacillaris Cori mutant W10BSmL, based on electron micrographs of serial sections of entire cells. Fluorescence due to normal plastid DNA is undetectable in these cells after treatment with the DNA fluorochrome 4'6-diamidino-2-phenylindole (DAPI). Serial sections through a newly described compartmentalized osmiophilic structure in Euglena cells are presented.     

Electron Microscope Autoradiography of DNA Synthesis in the Replication Band of Two Hypotrichous Ciliates1

The synthesis of DNA in two hypotrichous ciliates, Styx sp. and an amicronucleated strain of Oxytricha sp., was studied by high voltage (1000 kV) electron microscopy. High voltage EM permits use of thick sections (0.25-0.40 μm), including serial sections; thick sections produce strong autoradiographic images with relatively short exposure times. The autoradiographs show that DNA synthesis occurs in a narrow part of the rear zone of a replication band in the macronucleus. Macronuclear DNA synthesis occupies a substantial part of the interdivision interval, and micronuclear DNA synthesis in Styx sp. takes place in early prophase at a time when macronuclear DNA synthesis is in its terminal phase.     

Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.     

Coralline shape of the bacterial nucleoid after cryofixation.

A new procedure of immunostaining sections of cryofixed and freeze-substituted Escherichia coli shows that DNA extends from its bulk into small ribosome-free spaces throughout the cytoplasm, resulting in a coralline-shaped nucleoid. Low-resolution imaging of a bacterium reconstructed from serial sections demonstrated that the small excrescencies are not resolved. The resulting photograph shows the same features as phase-contrast light micrographs.     

DNA polymerase activity and DNA synthesis in roots of pea (Pisum sativum) seedlings

Soluble DNA polymerase (DNA polymerase-α) and chromatin-bound DNA polymerase (DNA polymerase-β) have been assayed in serial sections cut from the roots of 5-day-old pea seedlings. The activity of DNA polymerase-α is high in regions of the root which exhibit high rates of DNA replication, and declines during cell differentiation and maturation. The activity of DNA polyrnerase-β is low in cells which show high rates of DNA replication, and increases during differentiation and maturation.     

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 %.     

Video-enhanced technique for detecting neurophysin, enkephalin, and somatostatin immunoreactivity in ultrathin sections of cat median eminence

A freeze-drying technique using epoxy-embedded ultrathin serial sections permits critical comparisons of neuropeptides in small fibers and varicosities of the nervous system by video-enhanced, light microscopic immunofluorescence. The desirability of the method was documented by data showing: retention of radioimmunoassayable somatostatin in freeze-substituted blocks of tissue as compared to its loss in tissue dehydrated in an alcohol series; feasibility of OsO4 vapor fixation of freeze-dried tissue and compatibility with neuropeptide immunocytochemistry, and utility of a silicon-intensified-tube video camera for recording low levels of fluorescence from ultrathin sections. Ultrathin serial sections, 150 nm thick, from the inner zone of freeze-dried median eminence of the cat revealed three populations of axons containing various combinations of neurophysin immunoreactivity and enkephalin immunoreactivity. Some elements contained neurophysin immunoreactivity alone, some contained both neurophysin immunoreactivity and enkephalin immunoreactivity, and a few elements contained enkephalin immunoreactivity alone. The adjacent external zone of the median eminence contained immunoreactivity for all three substances, but the structures in this region were too small to permit demonstration of coexistence in 150 nm thick sections.     

Three-dimensional architecture of the higher plant nucleolonema disclosed on serial ultrathin sections

Summary— The three-dimensional architecture of the nucleolonema of Vicia faba has been studied by applying a silver impregnation technique to serial ultrathin sections. This technique disclosed lateral and transverse segments of the nucleolonema which were heavily impregnated with silver. The lateral profiles of the nucleolonema segments were classified into three main categories; a segment made up of one to several rod-like filaments (type I); a ladder-like segment consisting of two parallel and of transverse filaments (type II); and a last type constructed from two parallel filaments (type III). Tracing of the lateral segments through serial sections has indicated that type I first appears, then either type II or III and finally type I reappears at the corresponding sites on sections. Types II and III remained constant in width, about 1.0 μm, along their longitudinal axes whereas the width of type I was significantly smaller than that of the two former. The lateral filaments of both types II and III showed heterogeneity in width on account of the presence of knobs intermittently distributed along them. The thickness of these knobs was about 0.35 μm. Combining the observations on serial ultrathin sections and the morphometrical data it is very probable that the elementary structure of the nucleolonema is a 0.35-μm thick filament that tightly coils up into a solenoid structure with a thickness of approximately 1.0 μm. This model can explain the appearance of open- and closed-argyrophilic rings in serial sections since transverse segments of the solenoid are expected to show the argyrophilic rings. The elementary filament of the nucleolonema solenoid was sometimes loosened. Judging from our cytochemical data at the electron microscope level, some argyrophilic proteins appear to reside in the axial space of the solenoid but both DNA and RNA were not detectable in this space.     

Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence-independent amplification of DNA

A method that permits the in vitro amplification and cloning of DNA dissected from specific regions of a chromosome and does not require prior knowledge of the DNA sequence is described. DNA from several different chromosomal loci in the Drosophila melanogaster genome has been isolated by this method. Although the procedure was developed to permit the isolation of DNA sequences from serial sections of a single microdissected polytene chromosome, it should be useful for obtaining DNA clones from specific regions of the nonpolytene chromosomes of other organisms as well.     

A stereotaxic atlas of the monkey frontal lobes.

A stereotaxic atlas of the frontal lobes of M. Arctoides is presented as a consecutive series of line drawings. It is based on six animals. Alternating serial coronal sections stained with cresyl violet or Luxol Fast Blue were produced for each brain. Representative sections were used to prepare line drawings at one-millimeter intervals at a magnification of 4x. The stereotaxic anterior to posterior range is +43 to +20. This line drawing format can be use to recorded electrode placement or document the extent of lesions.     

LOCALIZATION OF CELL DIVISION ACTIVITY IN THE PRIMARY THICKENING MERISTEM IN ALLIUM CEPA L

Stems 1, 2, 3 months old of Allium cepa L. were labelled with tritiated thymidine, fixed in FAA, sectioned, stained with the Feulgen reaction, and prepared for autoradiography. The serial transverse sections were outlined with a camera lucida, recording labelled nuclei as dots. These drawings were used for 3-dimensional reconstructions of the locations of labelled nuclei. Near the top of the stem, labelled nuclei occur in a broad band, whereas they occur in narrower bands at successively lower levels in the stem, and finally labelled nuclei disappear. The locations of the labelled nuclei correspond to the location of the primary thickening meristem (PTM) in the stem of onion as determined by previous histological and histochemical observations. Microspectrophotometry was used to measure the relative amounts of DNA in Feulgen-stained nuclei of the PTM in serial transverse sections of 1- and 2-month-old onion stems. A bimodal distribution was obtained which can be explained by changes in DNA levels during the cell cycle. No evidence of polyploid nuclei was observed. One can conclude, therefore, that the PTM is the site of cell division activity during the primary stem thickening process in onion.     

Bulk Handling Racks for Free-Floating Serial Sections, Especially Suitable for Nauta Staining

Staining racks, each containing 20 shallow compartments, were constructed by drilling 1.5 cm holes in 8 × 11 cm sheets of 1 mm thick Darvic, an unplasticised polyvinyl-chloride compound, and cementing fibre-glass gauze of 1.3 mm mesh size to one surface. Sections were placed serially—one to each compartment—in the racks, and stacks of up to 9 racks were clamped by Perspex (methyl methacrylate) nuts and bolts, and side clamps. Thus, sections could be handled easily and kept in strict serial order, even in bulk. For Nauta staining, brains had to be gelatin embedded before sectioning. By storing sections in groups of 4 in ice-cube trays, evenly spaced series could be selected for placement in the racks. As many as 160 sections were taken together through all stages of the Nauta method, the timing of critical stages being controlled by taking 4 to 6 free-floating sections, together with the racks, through the various solutions.     

Mucosal nerves and smooth muscle relationships with gastric glands of the opossum: an ultrastructural and three-dimensional reconstruction study

The precise anatomical relation by which autonomic nerve endings contact gastric epithelial cells to enhance the rate of gastric secretions is not fully understood. The aim of the present study was to clarify this issue by using the technique of serial section reconstruction of areas of the gastric mucosa. The work also explored the possibility of a functional role for a system of smooth muscle strands in the gastric mucosa that emanate from the muscularis mucosa, run in the lamina propria, and are associated in a unique manner with the gastric glands. Electron microscopic serial sections of the gastric mucosa were performed to visualize the entire limiting membrane of gastric epithelial cells to determine any nerve associations (especially varicose endings) with these cells. Evaluation of serial sections of five separate parietal cells showed that their basal membrane did not come in close contact (nearest distance 500 nm) with any nerve axon or varicosity. Moreover, the axons passing in the area of these cells ultimately showed varicose endings associated with smooth muscle cells in the adjacent connective tissue (often separated by only 20 nm), with mast cells or with vascular elements. Additionally, the lateral membrane of these five parietal cells did not contact any endocrine cell in the epithelium, although other parietal cells in the area were adjacent to endocrine cells. Chief cells in the immediate area also did not form any close associations with nerve varicosities. Random analysis of 5,000 additional epithelial cells in these sections showed no close associations to nerve elements with significant accumulations of neurosecretory vesicles (varicosities). Because of the observed existence of innervation to the smooth muscle strands in the area of the gastric glands, serial 1-micron epoxy sections of the gastric mucosa were prepared, and profiles of smooth muscle and gastric glands were entered into a computer-assisted reconstruction system. Three-dimensional reconstruction techniques were employed to reveal the existence of a unique association between the mucosal smooth muscle strands and the gastric glands. The muscle strands arose from the muscularis mucosa at regular intervals and became branched to form an intricate wrap around a series of gastric glands that empty into one gastric pit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructure of holokinetic mitotic chromosomes and interphase nuclei of Tetranychus urticae Koch (Acari,Tetranychidae) in relation to loss and missegregation of induced fragments

The association of microtubules with mitotic holokinetic chromosomes of Tetranychus urticae Koch was investigated in serial ultrathin sections. Reconstructions from 14 series showed that 60–100 microtubules were associated with the entire poleward surfaces of the chromosomes. In the telophase of early developmental stages the chromosomes were decondenseed into separate micronuclei, containing at least one nucleolus. From these morphologic data, the fate of induced chromosome fragments, described in earlier papers, is surmised to depend on events in interphase as well as in mitosis.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

Interodontoblastic collagen (von Korff fibers) and circumpulpal dentin formation: an ultrathin serial section study in the cat

The collagenous fibers of von Korff pass from the dentin matrix between the odontoblasts into the dental pulp. Although collagen fibrils are known to be present between odontoblasts, the existence of von Korff fibers has remained controversial. This may be because their continuity between the dentin matrix and the pulp has not been demonstrated ultrastructurally. In this study we have examined the odontoblast layer in the middle to apical regions of perfusion-fixed permanent canine teeth of cats by using transmission electron microscopy. Ultrathin sections of demineralized specimens revealed frequent bundles of collagen fibrils 1) entering the odontoblast layer from the predentin, 2) present between odontoblast cell bodies, and 3) passing from between the odontoblasts into the pulp. The question of continuity of these bundles from the predentin, across the odontoblast layer into the pulp was examined in ultrathin serial sections. Unbroken continuity of a collagen bundle from the predentin between the odontoblasts into the pulp was established in a reconstruction of one series of 22 serial sections and was very strongly suggested by a number of other series in which the numbers of available sections restricted their full visibility. This investigation has shown, therefore, that classical von Korff fibers are present and that these fibers are present in fully erupted teeth with closed apices, i.e., at a time when secondary circumpulpal dentinogenesis is in progress. The findings call for a reexamination of the question of von Korff fibers during mantle dentinogenesis and primary circumpulpal dentinogenesis. Resolution of their existence at the earlier stages of dentinogenesis should be possible by using the ultrathin serial-sectioning technique.     

Risk biomarker assessment for breast cancer progression: replication precision of nuclear morphometry.

Nuclear morphometry is a method for quantitative measurement of histopathologic changes in the appearance of stained cell nuclei. Numerous studies have indicated that these assessments may provide clinically relevant information related to the degree of progression and malignant potential of breast neoplasia. Nuclear features are derived from computerized analysis of digitized microscope images, and a quantitative Feulgen stain for DNA was used. Features analyzed included: (1) DNA content; (2) nuclear size and shape; and (3) texture features, describing spatial features of chromatin distribution. In this study replicated measurements are described on a series of 54 breast carcinoma specimens of differing pathologic grades. Duplicate measurements were performed using two serial sections, which were processed and analyzed separately. The value of a single feature measurement, the nuclear area profile, was shown to be the strongest indicator of progression. A quantitative nuclear grade was derived and shown to be strongly correlated with not only the pathologic nuclear grade, but also with tubule formation, mitotic grade, and with the overall histopathologic grade. Analysis of replication precision showed that the standard methods of the histopathology laboratory, if practiced in a uniform manner, are sufficient to ensure reproducibility of these assessments. We argue that nuclear morphometry provides a standardized and reproducible framework for quantitative pathologic assessments.