Tissue fixation with phenol-formaldehyde for routine histopathology

Summary The addition of 2% phenol had a marked accelerating effect on neutral buffered 4% formaldehyde as a fixative. Histopathological material fixed in buffered phenol—formaldehyde (pH7.0) and rapidly advanced to paraffin in an enclosed tissue-processor showed improved nuclear and cytoplasmic detail, reduced shrinkage and distortion, and an absence of formalin pigment. Good results were obtained in less time when sequential fixation in phenol—formaldehyde buffered to pH7.0 and pH5.5 was carried out at an elevated temperature (40°C) in the enclosed tissue-processor. Standard histological stains and immunoperoxidase methods worked well. In resin-embedded tissue, buffered phenol—formaldehyde (pH7.0) gave satisfactory ultrastructural results. The penetration rate of buffered phenol—formaldehyde (pH7.0) in gelatin models did not differ from that of neutral buffered 4% formaldehyde. Polyacrylamide gel electrophoresis showed enhanced protein polymer formation with buffered phenol—formaldehyde (pH7.0) as compared with neutral buffered 4% formaldehyde. Protein polymer formation increased in response to increased time and temperature. Cells fixed in suspension in buffered phenol—formaldehyde (pH7.0) and neutral buffered 4% formaldehyde showed similar volume changes.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of Bombyx mori Linné were fixed in buffered (pH 8.2) 1% OsO₄ or 3 % KMnO₄ and thin sections of the tissue, embedded in methacrylate or epoxy Epon resin, were studied under the electron or light microscope.At the late stage of differentiation of the spermatid, the nucleus shows an elongated conical contour, being composed of fine fibrillar elements. These fibrillar elements fixed in OsO₄ measure 100 to 130 Å in diameter, while those fixed with KMnO₄ are approximately 70 Å in diameter.It has been found for the first time in the spermiogenesis of the silkworm that two bands and a tubular structure develop in close proximity to one another and attached to the plasma membrane of the spermatid. The two bands fixed in OsO₄ are electron dense, but in the material fixed with KMnO₄, one of them, situated within the cell body, is as dense as that fixed in OsO₄, while the other, outside the cell body, is much less dense. These apparently novel apparatuses develop from the caudal nuclear region along the elongating spermatid, but the dense band intertwines with the acrosome in the apical region of the nucleus along the major axis of spermatid, while the tubular structure and the clear band reach far into the nutritive cell where the dense band and acre-some are not visible.A possible relationship between the tubular structure and the nutritive cell has been discussed.This study was supported by Grant GM-8327-03 from the United States Public Health Service.     

A comparative electron microscope study of visceral muscle fibers in Cambarus,Drosophila and Lumbricus

Summary The arrangement of myofilaments in the striated visceral muscle fibers of two arthropods (crayfish and fruitfly) and in the unstriated visceral fibers of one annelid (earthworm) was studied comparatively. Transverse sections through the A bands of arthropod visceral fibers indicate that each thick myofilament is surrounded by approximately 12 thin filaments. The myofilaments are less organized in the visceral fibers of the earthworm than in muscle fibers of the crayfish and fruitfly. The thick myofilaments of the earthworm are composed of subunits, 20–30 Å in diameter. The presence of two distinct sets of myofilaments in these slowly contracting striated and unstriated visceral muscle fibers suggests that contraction is accomplished via a sliding filament mechanism.In crayfish visceral fibers the sarcolemma invaginates at irregular intervals to form a long and unbranched tubular system at any level in the sarcomere. Dyads formed by the apposition of T and SR membranes are observed frequently. The distribution of the T and SR systems in the visceral fibers of the fruitfly and the earthworm is markedly reduced and dyads are infrequently observed. The reduced T and SR systems may be related to the slow contraction of these fibers. Transport of specific substances across the sarcolemma could initiate contraction or relaxation in these fibers.This study was supported by a training grant GM-00582-06 from the U.S. Public Health Service.     

Electron-microscope studies of Drosophila salivary-gland chromosomes

Summary The submicroscopic structure of the chromosomes of the larval salivary gland cells inDrosophila melanogaster has been studied by means of electron microscopy of thin sections.The three fixatives used were buffered 1% osmium tetroxide, unbuffered Zenker's solution, and buffered formaldehyde in concentrations of 0·1% and 1·0%. It was found that formalin preserves the fine structure of the chromosomes better than the other fixatives.Nucleoprotein is present in the chromosome in the form of granules 200 Å to 300 Å in diameter. The granules are connected together by strands of protein approximately 100 Å in diameter. The length of the strands between successive granules in the banded areas is 100 Å to 300 Å and in the interband regions it is 700 Å to 900 Å.Heterochromatin differs from euchromatin in the absence or reduction in the amount of strand material.The nucleolus is composed of an aggregate of granules 300 Å to 600 Å in diameter contained in an amorphous matrix. Within the nucleolus are a series of canals containing material similar in appearance to that of the euchromatic band material.Submicroscopic granules are also present in the karyoplasm and in the cytoplasm surrounding the nuclear membrane. The cytoplasmic granules are more densely distributed near the nuclear membrane.A model for chromosome structure is proposed.The Applied Fisheries Laboratory is operated by the University of Washington under Contract No. AT(45-1)540 with the United States Atomic Energy Commission.     

A new interpretation of plasmodesmatal ultrastructure

Summary It is shown that simple, unbranched, plasmodesmata between young xylem ray cells of willow have no direct intercellular continuity apart from the plasmalemma which limits the cytoplasm and lines the plasmodesmatal canal. Each plasmodesma is traversed by a 200 Å diameter tubule (the desmotubule) which has a wall with probably 11 subunits arranged around a central cavity through which runs a 40 Å diameter rod. This rod is connected to the inside of the tubule wall, by fine filaments. At the ends of each plasmodesma the plasmalemma and cell wall are closely appressed to the tubule, thus precluding direct continuity between the cytoplasm of adjacent cells. Through the central part of the plasmodesmata the tubule is separated from the plasmalemma by a 90–100 Å wide gap. Cytoplasmic microtubules in the same tissue have a diameter of approximately 250 Å and a wall probably composed of 13 subunits: both desmotubules and cytoplasmic microtubules therefore have a centre-to-centre subunit spacing of about 47 Å. It is suggested that the desmotubules are not microtubules but may be nuclear spindle fibres which become trapped in the wall during cell plate formation. The endoplasmic reticulum, while closely approaching the plasmodesmata, is not continuous across them. It is thought most unlikely that the endoplasmic reticulum traverses plasmodesmata, as the dimensions of the central tubule — found here as well as by other workers — are smaller than those which would be expected to allow a stable molecular configuration in a unit membrane. The plasmalemma, where it lines the plasmodesmatal canal, appears to have particulate subunits in the outer opaque layers and the presence of these subunits may be attributable to the need for stability in membranes arranged about so small a radius.     

Use of nitrogen mustard N-oxide for the fixation of electron microscopic tissues

Summary Nitrogen mustard N-oxide was tried for the fixation of tissue for electron microscopy. A fixative consisting of 1% nitrogen mustard N-oxide, 1% glutaraldehyde and 1% paraformaldehyde buffered at pH 7.4 followed by 1% O_sO₄ buffered at pH 7.4 was found useful for the tissues examined: thyroid, anterior pituitary, adrenal gland and oviduct of mice.If the tissues are fixed and the sections are stained with uranyl acetate and lead acetate doubly, the follicle colloid, colloid droplets, and secretory granules containing thyroglobulin in the thyroid become higher in electron density. The cisterna of the maturing face of the Golgi apparatus, secretory granules, ribosomes, nucleolus and chromatin in the cells examined are extremely electron dense. Tubular elements of smooth endoplasmic reticulum in the adrenal cortical cell and microtubules in all the cells examined are also well preserved. The fixative containing nitrogen mustard N-oxide is useful also for cytochemistry. Using tissue fixed by this method and stained en bloc by uranyl acetate, the noradrenaline and adrenaline cells in the adrenal medulla are clearly distinguished by light microscopy.This study was supported by a grant from the Japan Educational Ministry     

Complete Staining of Neuromuscular Innervation with Bromoindigo and Silver

Frozen sections of musde fixed in buffered formaldehyde (pH 7.3) are first incubated for localization of esterase using 5-bromoindoxyl acetate as substrate. The sections are then mounted on slides and stained by a urea-silver nitrate method for axons. Result: subneural apparatus—blue; axons—black; other tissue components in various shades of grey.     

Application of cryo-ultramicrotomy techniques to microtubules of bracken (Pteridium aquilinum L.) sperm cells

Summary Mature antheridia from agar cultures of prothalli of the bracken fern were fixed for short periods of time in buffered 1% glutaraldehyde. Cryosections were obtained after rapid freezing in glycerol. In oblique cryo-sections through flagella rows of small globular subunits were observed making up each axoneme, the approximate size of each globule was 4.5 nm. Other superficial material was also found in the same sections. In transverse sections through the layer of microtubules which surround the helical nucleus, subunits of the order of 4.5 nm were observed.     

Nuclear and cell division inSorodiplophrys stercorea,a labyrinthulid-like protist

Summary Nuclear division in the labyrinthulid-like protist,Sorodiplophrys stercorea was studied and compared to the mitotic processes reported in other protistan forms.S. stercorea was found to have centrioles with nine peripheral singlet microtubules and a tubular core 300–400 Å in diameter. The nuclear envelope remains intact throughout division, with microtubules passing through nuclear pores. The nucleolus disappears during nuclear division. After intranuclear division, cells cleave by introgressive cleavage while pronucleoli appear in the nuclei and fuse to form the single nucleolus characteristic of the interphase nucleus.     

Studies on the fine structure of teleost blood cells

Summary Electron microscopic study of nucleated erythrocytes of the goldfish, Carassius auratus, reveals the microtubular elements comprising the marginal band which encircles the cell. Six to ten units are visible at each pole of the cell, immediately within the plasmalemma. Each tubular unit is composed of an electron dense membrane enclosing a less dense core. Cross-sectional units average 264 Å outer diameter, whereas tubules measured in longitudinal sections average 237 Å.The functions of the microtubules of the marginal bands are analyzed in view of Meves' original interpretation of maintenance of the discoidal form of the nucleated erythrocyte, and the more recent investigations in cell physiology of Trotter and Tilney. It is proposed that the microtubules possess a dual function: the support of the cell which is attributed to the hydroelastic properties of the turgid microtubules resulting from intratubular hydrostatic pressures; and the intracellular transport of materials via the intratubular fluid. The microtubules may, therefore, be considered as a skeletal system and part of an intracellular circulatory system.This project was supported by grants 2 G-895 and 2 G-505 from the United States Public Health Service.     

STUDIES ON NUCLEI USING CORRELATED CYTOCHEMICAL, LIGHT, AND ELECTRON MICROSCOPE TECHNIQUES

In this paper, a procedure for correlating electron microscope and light microscope cytochemical studies using immediately adjacent serial thin and thick sections has been described and discussed. This technique, combined with the Feulgen reaction for DNA, has been of particular value in framing and answering both general and specific questions about the nucleus. The results may be summarized as follows:— Apparent nuclear homogeneity in the electron microscope is not due to loss of DNA as evidenced by positive Feulgen reactions in such nuclei. Arrangement of Feulgen-positive material in chromosomes, heterochromatin, perinuclear and perinucleolar chromatin, etc., is similar to that customarily observed in the light microscope but this is not necessarily reflected in a cursory survey of the electron image. Careful comparison of light and electron images shows that fine differences in structure are associated with chromatin localization. Primary spermatocyte prophase chromosomes of crayfish have been positively identified by their Feulgen-positive nature. Core-like axial structures in such chromosomes have been observed (9) and are described further. A remarkable feature of spermiogenesis in the crayfish is an elaboration of the nuclear envelope of the spermatid accompanying the formation of what becomes a mass of convoluted membranes in the sperm. In the spermatid, perinuclear chromatin follows outpocketings of the nuclear envelope into the cytoplasm. In the early sperm, on the other hand, although the nuclear envelope is continuous with the system of convoluted membranes, the chromatin is distinct from it and is retained in the nucleus proper by some mechanism independent of the nuclear envelope. None of the above observations was apparent from the electron microscope images alone; they were possible only by virtue of the correlated cytochemical and electron microscope study of adjacent sections. The successful use of other cytochemical tests, such as the PAS reaction for certain carbohydrates, in such correlated studies is also described.     

Synaptology of the claustrum in the rat

A great deal of information is available on the morphology of the claustrum in various animal species, as well as on its neuronal distribution and relationships with the cerebral cortex and other nuclei. However, no research has been performed on synaptic organization. Here we report an ultrastructural study performed on 7 male albino rats of the Wistar strain weighing 270-310 g. Five rats were sacrificed by prolonging general anesthesia with diethyl ether until death. Three of these rats were secured to the stereotaxic atlas coordinates of Paxinos and Watson (1982); the claustrum area was marked by injecting 1 microliter of a 10% Evans Blue solution into the nucleus. The brain was then removed from the skull, cut into 2-3 mm thick coronal sections, and tissue samples taken from the area immediately adjacent to the marked area were immersed in 2% OsO4 buffered with 2% potassium dichromate containing 0.2% CaCl2 at pH 7.7 (Gobel, 1968). After dehydration they were embedded in Durcupan and the ultrafine sections were stained with uranyl acetate and lead citrate and observed with either a Zeiss 9S2 or a Hitachi H 800 electron microscope. The samples from two other rats, taken with the stereotaxic techniques described, were fixed for 12 h in 0.6 potassium permanganate solution buffered with veronal-acetate at pH 7.4 (Luft, 1956). After processing for electron microscopy, a portion of the sections were used without any contrast medium and the remainder were stained with uranyl acetate and lead citrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

MICROTUBULATION OF THE INNER MEMBRANE OF THE NUCLEAR ENVELOPE

In the course of a light and electron microscopy study of spermatogenesis in the European crayfish, Astacus fluviatilis, spermatocytes of abnormal appearance were observed in two instances in individuals that had passed the mating period. The electron microscope showed that the inner membrane of the nuclear envelope of these cells was erupting into a mass of microtubules, 15 to 18 mµ in diameter and 0.5 µ or more in length, while the outer membrane transformed into cytoplasmic vesicles. Stages in the formation of these novel processes were followed. The plasma membrane of the affected cells was seen in some cases to erupt into similar although shorter microtubules. It is concluded that the phenomenon is part of a degenerative process in which the spermatocytes are being absorbed by sustentacular cells. It is suggested that the observations provide further evidence for a fundamental functional as well as a morphological similarity between the membranes bounding the nucleus and the plasma membrane.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Staining of interphase nuclei and mitotic figures in cultured cells with Alcian blue 8GX

Summary Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction or Heidenhain iron hematoxylin but without the latters’ length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Postmortem diagnostics of renal diseases from semithin sections.

For the light microscopic postmortem study of mostly glomerular renal diseases, in addition to the paraffin technique, 0.5 mu thick (semithin) sections from material fixed in buffered formaldehyde and embedded in methacrylate or Durcupan ACM were used. The method allows for eventual electron microscopic examinations. The semithin sections were stained with methylene blue combined with basic fuchsin, as well as with periodic acid-silver methamine. The method is not a substitution, but the supplementation of the paraffin technique and is suited for the clarification of numerous fine details: in some cases the exact diagnosis was made in this way.     

Immunocytochemical localization of nerve growth factor: effects of fixation

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.     

The endoplasmic reticulum of mung-bean cotyledons: Quantitative morphology of cisternal and tubular ER during seedling growth

The ultrastructure of the endoplasmic reticulum (ER) in storage parenchyma cells in the cotyledons of mung beans (Vigna radiata L.) was examined during germination and seedling growth. Two different methods were used to visualize the ER: thin (0.08 m) sections of tissue fixed in formaldehyde and glutaraldehyde and post-fixed with osmium tetroxide, and thick (1 m) sections of tissue fixed in buffered aldehyde and post-fixed with zinc iodide-osmium tetroxide (ZIO). Changes in relative amounts of ER were quantified by morphometry (stereology).The ER occurs in two forms: a cisternal form with associated ribosomes which can be seen at all stages from imbibition to cotyledon senescence, and a tubular form which initially has associated ribosomes. Stereoscopic images of thick sections of cotyledons of 2-day-old seedlings show that the tubular ER consists of a three-dimensional array of interconnecting tubules which have numerous connections with the cisternal ER. The network of tubules and cisternae extends throughout the cytoplasm enveloping the protein bodies. Germination and seedling growth are accompanied by a reduction in the total volume occupied by the ER. This reduction is the result of a preferential loss of tubular ER and occurs largely before protein mobilization. Cisternal ER decreases during the first 48 h of imbibition and seedling growth, but storage cells subsequently show an increase in cisternal ER just prior to and during the period of protein mobilization. Cisternal ER remains conspicuous during the last phase of reserve mobilization when starch is broken down and the cells are starting autophagy.Abbreviations ER endoplasmic reticulum - ZIO zinc iodide-osmium tetroxide This is the second in a series of papers on the endoplasmic reticulum of mung bean cotyledons. The first paper is referenced herein as Gilkes and Chrispeels (1980)     

Microtubules with 15 subunits in cockroach epidermal cells

Since Ledbetter and Porter (1964) described the 13 subunits which are visible in cross sections of negatively stained plant microtubules, subsequent observations have generally confirmed this number. By using Mizuhira's fixative composed of tannic acid and glutaraldehyde, it is easyto demonstrate the subunits of microtublules without optical reinforcement Cytoplasmic microtubules and sperm axonemes, fixed with Mizuhira's fixtive, similarly show 13 subunits (Mizuhira's and Futaesaku, 1971, 1972; Futaesaky et al., 1972; Tilney et al., 1973). This paper will describe a particular type of microtubule in insect epidermal cells fixed with the above fixative. The number of the subunits is found to be 15 in tranverse sections.     

The intranuclear mitosis of the ciliates Paracineta limbata and Ichthyophtirius multifiliis

Electron microscope studies on the premetaphase stages of micronuclear divisions of Paracineta limbata and Ichthyophtirius multifiliis showed that spindle material also exists during interphase. In the case of I. multifiliis scattered microtubule fragments persist in the nuclear space; in P. limbata the micronuclei contain a small paracrystalloid which is suggested to be microtubular protein. Wide microtubules, varying in diameter from 300 to 400 Å develop during intranuclear prophase near the nuclear envelope in both cases. There are good reasons to assume that they function as a kind of stem body during the enlargement of the surface area of the nuclear envelope. Later micronuclear prophase stages of both species show a some-what different development. In I. multifiliis, there are scattered groups of short microtubular segments, partly in parallel array, whereas in P. limbata the wide tubules are transformed into normal microtubules of 180–200 Å diameter. The nuclei of both species are similar at late prophase and prometaphase stages. Bundles of interpolar microtubules run between the chromosomes, and single microtubules, presumably induced by the chromosomes, cross them at different angles. The chromosome-induced microtubules appear a short time after the interpolars. At prometaphase stage all microtubules show a highly parallel arrangement and therefore it is suggested that chromosomal tubules reach their final polar orientation by the action of cross-bridges.