Kinematographische Dokumentation einer amitotischen Zellteilung

Summary Identification of argyrophilic cells, in pancreatic islets of normal rabbits, is accomplished by light and electron microscopy in osmium-fixed plastic-embedded tissues.Fixative, pretreatment and pH of silver nitrate solution were essential for the light microscopic study to reveal argyrophilic cells in osmium-fixed plastic-embedded pancreatic islet tissue. The best result was obtained with Dalton's osmium fixation and buffered silver nitrate methanamine solution at pH 9.O. The cytoplasmic granules of argyrophilic cells generally are densely packed but some of the cells show only sparse silver impregnated granules in the cytoplasm. Occasionally there are some non-argyrophilic granular cells in which, after silver impregnation, the cytoplasm appears clear. There are three kinds of cells in the pancreatic islets, i.e., argyrophilic granular cells, non-argyrophilic granular (clear) cells, and beta cells (situated centrally in the islet and stained light yellow in silver impregnated sections).The cells known as argyrophilic cells in light microscopy can be identified as alpha cells in electron micrographs by comparison of consecutive sections of the same cell.The author would like to express his appreciation to professor Roy C. Swan for his generous guidance.     

A Golgi-Electron Microscope Method for Insect Nervous Tissue

Golgi's light microscopic method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 21/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50μm sections am made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

A Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 2 1/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50 mum sections are made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

Light and electron microscopic study on the endocrine cells of the pancreas in a marine teleost,Fugu rubripes rubripes

Summary The pancreatic endocrine tissue of Fugu rubripes rubripes consists of numerous round principal islets (Brockmann bodies) of various sizes scattered around the gall-bladder. The endocrine cells are divided into A-, B-, D-, and F_f-cells. Each cell type was identified by comparing thick and thin sections in both light and electron microscopy. Aldehyde-fuchsin positive B-cells contain numerous round secretory granules (average diameter 300 nm) each of which has a round compact core of moderate density; a narrow space exists between this core and the limiting membrane. Grimelius' silver positive A cells contain round secretory granules (average diameter 360 nm) with a hexagonal or tetragonal crystalline core (average diameter 170 nm) of high density; the silver grains preferentially appear in the space between the limiting membrane and the core. The crystalline core of each -granule often contains an appendix-like structure of variable shape. D cells blackened by the silver impregnation method of Hellman and Hellerström (1960) have round secretory granules (average diameter 320 nm) filled with a flocculent material of low density. The fourth cell type (F_f-cell) has a clear cytoplasm after differential staining for light microscopy. By electron microscopy, this cell has elongated fusiform secretory granules (520 nm average length × 230 nm average width) filled with numerous filaments arranged in parallel with the longitudinal axis. Figures suggesting granule formation in the sacs of the Golgi apparatus were obtained in all of islet cell types. Equivalents of emiocytotic release of secretory granules were encountered in the A and F_f cells.     

Stabilization of Silver Chromate Golgi Impregnation in Rat Central Nervous System Neurons Using Photographic Developers. I. Light Microscopy

Deterioration of Golgi impregnation begins immediately after impregnated tissue blocks are sectioned with the Vibratome. The first signs of deterioration are fading of delicate impregnated processes, the disruption and fragmentation of dendrites, and, eventually, fading of entire neurons. These changes can be prevented by stabilization, i.e., by converting the water soluble silver chromate Golgi precipitate into metallic silver or by replacing the silver with some other dense, insoluble material. A technique is described using photographic developers to treat Vibratome sections containing Golgi-rapid or Golgi-Kopsch impregnated CNS neurons. In this way part of the silver chromate Golgi precipitate is reduced to metallic silver, and the remaining silver chromate is then removed with sodium thiosulfate. Of the various developers tested, Kodalith and Elon-ascorbic acid gave the best results, with excellent stabilization of the most delicate stuctures, such as the stalks of dendritic spines and finely woven axonal plexuses. Treatment with other developers (HC-110, Neutol, D-19, D-76, D-163, Kodak Universal, Rodinal, Atomal, Diafine, Eukobrom, Microdol-X) resulted in stabilization ranging from good to poor. Good stabilization of Golgi impregnation could also be achieved by first exposing the sections to sodium bromide (bromide substitution) followed by treatment with D-19, Kodalith, Elon-ascorbic acid or HC-110. After stabilization, the sections can be counterstained with aqueous cresyl violet or with alcoholic thionin without degradation of the stabilized Golgi image. The countentain permits exact determination of the position of impregnated neurons in cortical layers or subcortical nuclei.     

Stabilization of silver chromate Golgi impregnation in rat central nervous system neurons using photographic developers. I. Light microscopy

Deterioration of Golgi impregnation begins immediately after impregnated tissue blocks are sectioned with the Vibratome. The first signs of deterioration are fading of delicate impregnated processes, the disruption and fragmentation of dendrites, and, eventually, fading of entire neurons. These changes can be prevented by stabilization, i.e., by converting the water soluble silver chromate Golgi precipitate into metallic silver or by replacing the silver with some other dense, insoluble material. A technique is described using photographic developers to treat Vibratome sections containing Golgi-rapid or Golgi-Kopsch impregnated CNS neurons. In this way part of the silver chromate Golgi precipitate is reduced to metallic silver, and the remaining silver chromate is then removed with sodium thiosulfate. Of the various developers tested, Kodalith and Elon-ascorbic acid gave the best results, with excellent stabilization of the most delicate structures, such as the stalks of dendritic spines and finely woven axonal plexuses. Treatment with other developers (HC-110, Neutol, D-19, D-76, D-163, Kodak Universal, Rodinal, Atomal, Diafine, Eukobrom, Microdol-X) resulted in stabilization ranging from good to poor. Good stabilization of Golgi impregnation could also be achieved by first exposing the sections to sodium bromide (bromide substitution) followed by treatment with D-19, Kodalith, Elon-ascorbic acid or HC-110. After stabilization, the sections can be counterstained with aqueous cresyl violet or with alcoholic thionin without degradation of the stabilized Golgi image. The counterstain permits exact determination of the position of impregnated neurons in cortical layers or subcortical nuclei.     

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO₄ and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO₄ fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes.     

A Modified Silver Impregnation Technique for the Observation of Thick Sections of Lymph Nodes Perfused with Colloidal Carbon

For many years, a variant of the silver impregnation technique of Bielchowsky has been used to study the lymph node because it clearly outlines the various structures which are usually hard to contrast with standard staining methods. Like other variants of silver impregnation, this method blackens the cell nuclei as well as the reticular fibers; however, it inhibits the impregnation of the nuclear chromatin immediately adjacent to fibers. Hence, this variant selectively darkens the lymphoid cell populations of the nodal structures which contain a loose fiber network.

To study the blood vascular network of the lymph node based on perfusion of colloidal carbon, a staining procedure was needed which would contrast nodal structures on thick sections, while allowing the carbon-filled small blood vessels to be distinguished from the impregnated coarse reticular fibers. In an attempt to adapt this variant of Bielchowsky's technique, 10, 20, 40 and 60 nm thick sections from rat nodes, fixed in a solution of Bouin-Hollande for 72 hr, were silver impregnated with serial dilutions (1:2 to 1:128) of the ammoniacal silver solution. Forty-micrometer thick sections impregnated with a 1:16 dilution of the original silver solution at 37 C and for 30 min provided the best results for the conditions.     

Golgi impregnation with potassium dichromate and mercurous or mercuric nitrate: Identification of the precipitate by X-ray and electron diffraction methods

Summary Golgi impregnation of nerve tissue can be obtained by soaking the tissue in a solution of potassium dichromate followed by a solution of mercurous or mercuric nitrate. The precipitate formed in each of these two methods has been identified. Samples of impregnated tissues were ground and examined with X-ray powder diffraction and the patterns of spacings compared both with the values of international tables (the ASTM index) and with the results of X-ray studies of substances prepared by ourselves and subjected to quantitative chemical analysis. It was found that impregnated blocks contained mercurous chromate, Hg₂CrO₄, after mercurous nitrate treatment, and mercuric oxide chromate, Hg₃O₂CrO₄, after mercuric nitrate treatment. Selected area electron diffraction of ultrathin sections through impregnated nervous structures (embedded in epoxy resin) showed these to contain the substances demonstrated by X-ray analysis of whole tissue.This study was supported by U. S. P. H. S. Grant NS 07998. This aid is gratefully acknowedged. We are indebted to Mrs. E. Clausen, Mrs. K. Kirkholt, Mr. A. Meier and Mrs. K. Sörensen for technical assistance.Institute of Geology, Aarhus University, Aarhus, Denmark.We are indebted to him for the possibility of basing some of our studies on his results.     

Application of silver stains to gastric endocrine cells in plastic sections

Summary A simultaneous light and electron microscopic study of mouse gastric mucosa was made to determine whether the silver nitrate methenamine stain of Duk-Ho Lee could be used to stain gastric endocrine-like cells in plastic embedded tissue. Examination of consecutive thick and thin sections showed that this stain blackened the granules of the predominant type of endocrine-like cell present. Blackening of the granules with silver occured in tissue fixed in osmium tetroxide solution with or without dichromate salt or in tissue fixed in glutaraldehyde then treated with osmium. The intensity of staining was deepest in the osmium-dichromate fixed tissue, but the glutaraldehyde-osmium procedure gave less interference from diffuse silver impregnation and better preservation of detail for electron microscopy.     

LR White embedding allows a multi-method approach to the analysis of brain tissue from patients with Alzheimer''s disease

Summary Light (LM) and electron (EM) microscope comparisons of the cytochemistry and immunocytochemistry of neuritic plaques and neurofibrillary tangles, the main histopathological changes in the brains of Alzheimer's disease sufferers, have been almost impossible because of the disparity between the two technologies. By embedding unosmicated brain tissue in the acrylic resin LR White, direct comparisons can be made between techniques applied at the LM level with those at the EM level.After partial dehydration in 70% ethanol, the tissue is embedded by rapid infiltration and polymerization at 0°C, which has been shown to maximally preserve tissue immunoreactivity. Semithin sections are then receptive to routine LM stains, silver stains e.g. Gomori's methenamine silver, and immunocytochemistry with immunoperoxidase or immunocolloidal gold. Serial thin from the use of a mouse monoclonal antibody against -amyloid and a rabbit polyclonal antibody against ubiquitin are presented. LR White resin includes no elements other than carbon, oxygen and nitrogen, of which it is composed, so that sections of it are valuable for sensitive X-ray energy dispersive microanalysis.     

A simple impregnation technique for thin and semithin enterochromaffin cells in sections

Summary Constant, intense and precise impregnation of enterochromaffin (EC) cells was achieved simply by floating thin or semithin sections of gut mucosa, fixed in osmium tetroxide or in glutaraldehyde with postfixation in osmium, on a silver nitrate or proteinate solution. EC cells alone showed impregnation in the light microscope. In the electron microscope, impregnation affected not only the secretory granules of EC cells but also, although much more faintly, those of other, non-EC cells (D, X, D₁, G and other cells). Lysosomes also showed partial or total reactivity. Oxidation reduced but did not entirely suppress EC cell staining and had no effect on non-EC endocrine cell staining. Since the reaction did not occur with glutaraldehyde alone, osmium appeared to be a crucial component of the process. These findings should be borne in mind in applying Thiery's method for vicinal glycol groups to the type of study material used in these experiments.     

Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of P 0 mRNA in myelin-forming Schwann cells

Summary A biotinylated P ₀ glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelinforming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain P ₀ mRNA. Interestingly, P ₀ mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

A Method for Silver Impregnation of Mammary Myoepithelia

Histochemical methods for microscopic visualization of nummary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Morphology and histochemistry of the adrenal medulla

Summary Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections.Four types of primary catecholamine-storing cells were identified. NA₁ cells contain cytoplasmic granules up to 0.3 m in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA₂ cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA₃ cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA₄ cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence.The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA₁ cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA₂ cells correspond to this latter population (175 nm). NA₃ cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA₄ cells being in the dimension of 80 nm. Granules in NA₁ and NA₂ cells show uniformly high electron density, whereas those in NA₃ and NA₄ cells display cores of varying density. Granules with moderately dense cores in NA₃ and NA₄ cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.Supported by the Deutsche Forschungsgemeinschaft, grant Nr. Bo 525/1These results were presented in part at 17. Tagung der Deutschen Gesellschaft für Elektronenmikroskopie, Berlin, 21.–26. 9. 1975     

A method for silver impregnation of mammary myoepithelia

Histochemical methods for microscopic visualization of mammary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Golgi potassium-dichromate silver-nitrate impregnation

Summary Golgi preparations were made by consecutive treatment of formalin-fixed brain and liver with potassium dichromate and silver nitrate. Impregnated tissue dissected from thin slices of the blocks were studied by X-ray powder diffraction methods, in a diffractometer and a Guinier camera. Such tissue proved to contain crystalline silver chromate, Ag₂CrO₄, both while still in the silver nitrate solution and after dehydration in ethanol and clearing in xylene and xylene-Dammar resin. No other compounds containing chromium or silver were detectable. Formalin-fixed tissue merely treated with silver nitrate contained silver chloride, but in impregnated tissue the amount was too scarce to be visible. Hence, silver chloride was no integral part of the Golgi precipitate.A number of mostly ethereal oils traditionally used for clearing histological sections, did not cause the appearance of metallic silver in detectable amount in the Golgi preparations. However, after treatment with clove oil and creosote metallic silver was detected in the tissue.This study was supported by U.S. P.H. S. Grant NS 07998. This aid is gratefully acknowledged.We are indebted to Miss I. Madsen and Mrs. K. Sörensen for technical assistance.     

Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat,chick and human)

Summary Explants of embryonic and fetal spinal cords (rat, chick, and human) develop and maintain in vitro many cytologic and bioelectric properties characteristic of central nervous tissues in situ. Despite the thickness of the cord explants, a condition which appears to be necessary for differentiation, considerable neuronal development becomes visible to light microscopic examination.The mature expiant consists of large concentrations of small neurons interspersed with large neurons and neuroglia, bounded by a broad tract of nerve fibers and capped by a neuropil. Myelination in rat cultures usually begins 2–3 weeks after explantation in both the explant and outgrowth zone. Myelin is of the central glial type unless the cord explants are grown with their meningeal covering. In the latter case the myelination pattern abruptly changes to the peripheral Schwannian type as the axons penetrate the meninges.Bouton-like endings are observed in the neuropil of rat cord explants (whole-mounts) impregnated with silver; but most neurons are only partially blackened. In 20 sections, neuronal somas and dendrites are identified in negative image with blackened bouton-like endings suggesting synapses.Chick spinal cord, when grown in Rose chambers, becomes more thinly spread so that more detailed interrelationships can be visualized in the living neuronal somas, neuritic processes and termini. Bouton-like endings on neuronal somas have been selectively stained, vitally, with methylene blue.Complex bioelectric activity can be evoked in these long-term spinal cord explants by electric stimuli localized to various regions of the cord tissues as well as to attached dorsal-root ganglia. The long-lasting after-discharge patterns and the neuropharmacologic sensitivity of the responses show remarkable similarity to the activity of synaptic networks of the central nervous system in situ. These functions develop gradually during the first week after explantation of fetal rat cord tissues — more slowly in cultures explanted before the establishment of reflex arcs in utero, and more rapidly in cord explants from older fetuses. Reference is made to the companion electron microscopy study of older fetuses, which shows that characteristic synaptic structures, although extremely rare at the time of explantation, are abundant in later stages of the culture's development. This confirms the functional evidence that synapses are able to develop in organized culture conditions.This study was supported by grants NB-00858 and NB-03814 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service.Research Career Development Fellow (Award NB-K3-2904 from the NINDB, USPHS).Research Career Award 5K6-GM-15,372 from U.S. National Institutes of Health.     

Chemical Stabilization of Golgi Silver Chromate Impregnations

Blocks of neural tissue were processed by a modified Golgi-Kopsch procedure and by the rapid Golgi method. Following the impregnation, the blocks were embedded in celloidin, sectioned at 100μm, and collected in 70% alcohol. The sections were then processed as follows: 1) rinsed in distilled water; 2) substituted with 0.4M sodium bromide for five minutes; 3) reduced in Kodak D-19 developer; and 4) treated in 0.5M sodium thiosulfate. The silver chromate deposits within the impregnated cells are converted successively to silver bromide and to reduced silver by this procedure. Sections so treated resist decomposition of the Golgi impregnation, and they may be counterstained with conventional aqueous cresyl violet to demonstrate the cytoarchitecture of the Golgi-impregnated tissue.     

Chemical stabilization of Golgi silver chromate impregnations.

Blocks of neural tissue were processed by a modified Golgi-Kopsch procedure and by the rapid Golgi method. Following the impregnation, the blocks were embedded in celloidin, sectioned at 100 micrometer, and collected in 70% alcohol. The sections were then processed as follows: 1) rinsed in distilled water; 2) substituted with 0.4M sodium bromide for five minutes; 3) reduced in Kodak D-19 developer; and 4) treated in 0.5M sodium thiosulfate. The silver chromate deposits within the impregnated cells are converted successively to silver bromide and to reduced silver by this procedure. Sections so treated resist decomposition of the Golgi impregnation, and they may be counterstained with conventional aqueous cresyl violet to demonstrate the cytoarchitecture of the Golgi-impregnated tissue.