A Five-Hour Variant of Gomori's Methenamine Silver method for Argentaffin Cells

Gomori's methenamine silver method for argentaffin cells has been modified and considerably accelerated by almost doubling the silver concentration and raising the incubation temperature to 60°C. Argentaffin cells are selectively impregnated in 3 to 4 hours and the background remains relatively clear up to 4 hours. The contrasts are clearer than with the ammoniacal silver methods of Masson and of Gluckmann.     

The fine structure of the proliferative cells of the mouse intestine as revealed by electron microscopic autoradiography with 3H-thymidine

Summary The duodenal and colonic epithelia in mice were observed with electron microscopic autoradiography 2, 5 and 24 hours after a single injection of ³H-thymidine. After 2 hours, in the duodenum, silver grains are found in many undifferentiated cells, in a few young goblet cells, in some crystal-containing cells, and in some lymphocytes. In the colon after 2 hours silver grains are seen in some undifferentiated cells, and in many young goblet cells. Undifferentiated cells are characterized by a few short microvilli, poorly developed rough-surfaced endoplasmic reticulum, abundant free ribosomes, and a few apical moderately dense granules. In normal animals, absorptive cells seem to arise from undifferentiated cells, and goblet cells — from younger goblet cells. Undifferentiated cells could also become young goblet cells. Crystal-containing cells, which may not be of epithelial origin, proliferate in the epithelium in the adult animal.     

Electron microscopic radioautographic study on the incorporation of 3H-proline by mouse decidual cells

A qualitative radioautographic analysis showed that mature decidual cells of the mouse are able to incorporate 3H-proline. After 1 hour silver grains are concentrated mainly over these cells although some of them can be found over collagen fibrils. After 2,6 and 24 hours there is a progressive increase of silver grains on the extracellular space most of them concentrated over thick collagen fibrils. These results strongly indicate that mature decidual cells of the mouse produce collagen and that they conserve a behavior of the fibroblast from which they originated.     

Quantitative electron microscopic autoradiography on the mouse thyroid gland after administration of 3H-L-DOPA

Summary The cellular and subcellular distribution of radioactivity in the mouse thyroid gland different times (20 min — 8 hours) after intravenous administration of ³H-L-DOPA was studied by means of quantitative electron microscopic autoradiography.High concentrations of autoradiographic silver grains occur over parafollicular cells and adrenergic nerves while the labelling of follicular cells and lumina is low or absent and similar to the labelling of connective tissue cells at all observation times.Over the parafollicular cells high levels of radioactivity can be recorded already 20 min after administration of the labelled amino acid. The grain counts are highest at 1 hour and decrease then at 2.5 and 8 hours.The intracellular distribution of label is similar at all observation times; thus, the concentration of silver grains over the typical cytoplasmic granules of the parafollicular cells is 4–5 times higher compared to the concentration over the remainder of the cytoplasm and the nucleus.Treatment with a decarboxylase inhibitor prior to the injection of ³H-L-DOPA results in a low and uniform labelling of all thyroid cells. This finding, taken together with the observation that also pretreatment with reserpine abolishes the autoradiographic reaction over the cytoplasmic granules, gives strong support to the idea that the great majority of silver grains observed over parafollicular cells represents dopamine formed by decarboxylation of the labelled precursor.This study was supported by grant K71-12X-3352-01 from the Swedish Medical Research Council. The author wishes to express his gratitude to Mrs. Gunnel Bokhede and Miss Dala Sjögren for expert technical assistance.     

TSH stimulation of iodine organification in early foetal rat thyroid in vitro

Thyroid glands from 15 day-old rat foetuses were incubated in Eagle's medium containing Na 125I and supplemented, or not, with TSH for 4 or 24 hours. Electron microscopic radioautographic study shows silver grains mainly in follicular cavities only in the thyroids submitted to TSH during 24 hr. A functional differentiation must therefore take place in thyroid cells under TSH stimulation.     

建立小鼠膀胱肿瘤原位模型的优化方法

目的探讨硝酸银、盐酸、胰酶和乙醇预处理构建鼠膀胱肿瘤的成瘤机制。方法 24G静脉留置针插入膀胱,PBS冲洗后,将小鼠随机分为5组,每组6只:(1)乙醇作用组:22%乙醇0.1 mL保留20 min;(2)胰酶作用组:0.2%胰酶保留30 min;(3)酸碱作用组:0.1 mmol/L HCl 0.1 mL作用15 s后,PBS冲洗,0.1 mmol/L NaOH0.1 mL作用5 s,排空膀胱;(4)硝酸银作用组:0.15 mol/L硝酸银保留10 s;(5)对照组:0.1 mL生理盐水。术后1和24h随机处死每组3只小鼠,摘取膀胱,HE染色观察膀胱黏膜病理变化;戊二醛固定,电镜下观察膀胱黏膜细胞微结构变化;甲苯胺蓝染色,观察膀胱黏膜固有层肥大细胞数目变化;过碘酸-希夫(PAS)染色,观察膀胱黏膜GAG层变化。40只小鼠应用上述前四组预处理因素处理膀胱后,建立膀胱癌原位模型,计算各组成瘤率。结果胰酶和乙醇处理1h后,局部上皮伞状细胞脱落,黏膜下层暴露;酸碱和硝酸银处理组大部黏膜完整性破坏,黏膜下层暴露较多,连续性中断;对照组和实验组间炎症细胞浸润均不表现出统计学差异。24 h后,胰酶和乙醇组可见局部轻度水肿并充血,黏膜完整性恢复较好,细胞间见紧密连接;而酸碱和硝酸银组上皮黏膜薄厚不均一,仍可见部分脱落黏膜。结论利用硝酸银和酸碱预处理膀胱可作为鼠膀胱肿瘤原位模型构建的首选方法。     

Staining Pineal Parenchyma by A Modified Hortega Method After Paraffin Embedding

Fresh pineal glands are fixed in 10% formalin at room temperature for about 3 days. After washing, dehydrating and clearing they are embedded in paraffin, sectioned and mounted. The tissues are placed in 10% silver nitrate for 24 hours, washed and impregnated in strong silver carbonate. The sections are reduced in 10% formalin, washed and toned in gold chloride, fixed in 5% hyposulfite, counterstained with erythrosin and mounted in Canada balsam. The processes of the pineal parenchymatous cells of the sheep, cow and man have been successfully stained by this method.     

Bodians Protargol Method Applied to Other than Neurological Preparations

Some applications of the Bodian technic for other than neurological purposes are described. After fixation for 48 hours, in formalin, 5 ml., acetic acid, glacial, 5 ml., and 80% ethyl alcohol, 90 ml., the routine procedures are recommended, with the exception that the exposure to protargol for 24 hours and subsequent reduction should be repeated once. Gold toning may be o-mitted. With this method the argentaffin (chromaffin) cells of the digestive tract (rat, hamster), the alpha cells of the pancreatic islands (hamster) and the medullary cells of the suprarenal gland (hamster) are selectively impregnated. In the mammalian pituitary gland (rat, hamster) certain of the granulated chromophile cells are impregnated. In the rat only the basophiles appear to react with the silver.     

Electron microscopic radioautography of intramitochondrial RNA synthesis of HeLa cells in culture

Summary HeLa cells were cultivated in vitro and incubated in a medium containing ³H-uridine (20 c/ml) for 30 minutes 1, 2, 4, 8 and 18 hours, doubly fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, embedded in Epon or hydroxypropyl methacrylate, radioautographed with Sakura NR-H2 emulsion, exposed for 40 days, developed with gold-latensification and elon-ascorbic acid developer.As the results, silver grains appeared not only in the chromatin, nucleoli, endoplasmic reticulum and ribosomes but also in the mitochondria. Most of the silver grains found in the mitochondria were localized on the mitochondrial matrix, while some others were on the cristae and the mitochondrial membranes. These silver grains were removed with RNase digestion. The percentage of labeled mitochondria increased in accordance with the time of incubation. Almost all the mitochondria were labeled within 18 hours.From the results, it was concluded that the mitochondria synthesized RNA at their matrices.     

Cupular secretion by Xenopus laevis line organs: autoradiographic evidence for incorporation of 3H-glucose and 35S-sulfate

Autoradiographic evidence for incorporation of 3H-glucose and 35S-sulfate into the cupulae of Xenopus laevis (African clawed toad) lateral line organs was obtained after injection into the dorsal lymph sacs of adult animals. Time intervals of 15 minutes to 4 hours after administration of these labeled metabolic precursors were used to examine the time course of the apparent mechanism of growth of the cupulae. Our results suggest that the two layers of accessory cells (the sustentacular cells and inner layer of mantle cells), concentrically arranged around the organ's central sensory (hair) cells, elaborate distinct cupular components. Sustentacular cells, immediately adjacent to the sensory cells, appear to produce and extrude at their exposed apices a cupular "core" substance labeled by 3H-glucose, but not by 35S-sulfate. The layer of inner mantle cells, external to the sustentacular cells, was labeled by both precursors and is spatially situated to secrete a cupular sheath enclosing the cupular core. Ultrastructural differences between the secretory products within the two cell types were marked. Electron microscopic autoradiography of toads killed 4 hours after 3H-glucose injection showed that silver grains were associated with accumulations of the respective secretory products in sustentacular and inner mantle cells, and label was found over the cupular trough area, where the bases of the cupulae are attached. These results suggest that the cupular core and sheath may both contain mucopolysaccharide, and the sheath, a sulfated mucopolysaccharide.     

Electron microscopic autoradiography for demonstration of pineal serotonin in rat

Summary The fine localization of rat pineal serotonin has been studied by means of electron microscopic autoradiography. Two hours after the intravenous injection of tritium labelled 5-hydroxytryptophan, the location of large number of silver tangled threads is seen in the sympathetic nerve terminals. There is also a less specific accumulation of the silver grains in the pinealocytes, some appearing in the cytoplasmic organelles and some in the nucleus.In quantitative terms, 43% of the total count of silver grains were in the nerve endings whereas pinealocytes, which comprise a much larger volume of the section, contain a proportionally much smaller number of silver particles (53%). Furthermore the perivascular spaces, which comprise a larger percentage in volume of the section than the nerve endings has nevertheless only about 4% of the grains counted.Although the precise localization of the silver grains is obscure, the reaction of the granulated vesicles in the nerve terminals to the double fixation used, is similar to that shown by the extremely dense material in vesicles of platelets, which was demonstrated to contain serotonin. The results therefore suggest that the silver grains appearing in the nerve terminals two hours after the administration of 5-hydroxytryptophan are in the serotonin binding site in the axon terminals, containing the granulated vesicles.     

Silver activates calcium signals in rat basophilic leukemia-2H3 mast cells by a mechanism that differs from the Fc epsilon RI-activated response

We previously showed that silver stimulates degranulation and leukotriene (LT) C(4) production in rat basophilic leukemia mast cells and now show that silver induces these events by a mechanism that differs from the FcepsilonRI-mediated response. In common with FcepsilonRI cross-linking, silver induced tyrosine phosphorylation of extracellular signal-regulated kinases and furthermore, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinase dose-dependently inhibited the silver-induced LTC(4) production. In contrast to FcepsilonRI cross-linking, silver had no effect on the production of IL-4 and TNF-alpha, indicating that different mechanisms are involved in the activation by these two stimuli. In line with this, silver had no or only marginal effect on the tyrosine phosphorylation of FcepsilonRIbeta, Lyn, Syk, and linker for activation of T cells, the early and crucial events in FcepsilonRI signaling. Silver induced calcium signals that were involved in the metal-induced degranulation, but not LTC(4) production. Unlike Ag, the silver-induced calcium signals were resistant to the depletion of thapsigargin-sensitive calcium stores and the inhibition of tyrosine kinases and phospholipase Cgamma. These findings indicate that silver activates mast cells by bypassing the early signaling events required for the induction of calcium influx. Our data strongly suggest the existence of an alternative pathway bypassing the early signaling events in mast cell activation and indicate that silver may be useful for analyses of such alternative mechanisms.     

Visualization of Legionella Pneumophila in Paraffin Sections using Hexamine Silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 μm. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

Visualization of Legionella pneumophila in paraffin sections using hexamine silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 microns. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

An electron microscopic autoradiographic study of proline incorporation by mouse lingual epithelium

Summary Mouse lingual epithelium incorporates significant amounts of L-proline-2, 3-H³ one hour after intraperitoneal injection of the tritiated amino acid. All viable cell strata incorporated approximately equal amounts of proline as assessed by autoradiographic techniques. Grain counts at 30 minutes, 1 hour, 4 hours and 24 hours, the four time periods studied, indicated a progressive incorporation of proline up to 4 hours following injection. Preferential incorporation of proline into any one cell structure or group of structures was not observed. Keratohyalin granules (KHG's) demonstrated incorporated proline; however, usually only one silver grain appeared over each granule, and, based on grain counts, the amount of proline incorporated by KHG's appeared slightly less than the general labeling observed in KHG-containing cells. This finding supports recent biochemical studies which have indicated a considerably lower proline content of keratohyalin than had previously been reported. Significant proline incorporation into the epithelial basal lamina was not observed during the 24 hours of this study. Thus, while recent recombination experiments have conclusively demonstrated that epithelial basal cells synthesize considerable quantities of basal lamina in a 24 hour period; it would appear that epithelial basal cells contribute little to a formed, intact basal lamina. This finding lends credence to the concept of a long basal lamina turnover time.Supported by Public Healths Service grants DE 02731, DE 03393The authors are grateful to Dr. John H. Lillie for his help in determining blood levels of proline-H³ and to Dr. V. C. Hascall for his advice on isotope selection. Mrs. K. Y. Y. Chen performed nearly all technical matters associated with this study, and made many of the original electron microscopic observations. Her assistance was invaluable.     

Construction of a very high-density extracellular electrode array

Cellular activation mapping (specifying in time and space the electrical activation sequence of cells) is a well-established basic research tool in cardiac, neural, and gastric physiology. Much recent research in cardiac mapping has focused on large arrays (>200 electrodes) with small electrodes (<500 microm). Construction of such arrays using standard techniques is tedious and yields irregular electrode spacing. We present a novel construction technique that rapidly produces large arrays with regularly spaced small electrodes. For methods, fine-pitch copper ribbon cables, insulated with either polyvinylchloride (PVC) or polyimide (flexible printed circuit; FPC), were assembled together such that the active surface was the cut end of the cable. The cut end was sanded and polished, then coated with silver and sometimes silver chloride. Once completed, the alternating current (AC) root-mean-square (rms) potential was measured between two adjacent, individual electrodes. Polarization testing was conducted according to a previously reported protocol (Witkowski FX and Penkoske PA. J Electrocardiol 21: 273-282, 1988). Activation mapping was conducted in the open-chest guinea pig with both pacing- and defibrillation- strength stimuli. In terms of results, four PVC and three FPC arrays were constructed, ranging from 4 to 400 electrodes. Two hours of labor were needed to create a complete electrode array, independent of the number of electrodes, including connectors and silver/silver chloride coating. As expected, the addition of a silver/silver chloride coating significantly reduced (0.76-0.42 mV, P < 0.001) the AC rms potential difference between two electrodes. A nearly immediate recovery of the potential difference between adjacent pairs of silver/silver chloride electrodes was observed after defibrillation stimuli.     

Silver staining of the nucleolar organizer regions (NORs) in semithin Lowicryl sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH4Cl and Schiff's reagent prior to Ag-NOR silver staining.     

Silver Staining of the Nucleolar Organizer Regions (NORS) in Semithin Lowicryl Sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH₄Cl and Schiff's reagent prior to Ag-NOR silver staining.     

A Direct Silver Method for the Golgi Apparatus

Direct immersion of fresh tissue in a solution of silver in formalin at pH 4, followed by development in hydroquinone-formalin, results in consistent silvering of the Golgi apparatus. The time required depends on the penetration of the tissue, two hours for each step being adequate for routine purposes. Proper general fixation of the tissue is enhanced by returning it to a fixative for the customary periods of time. A weak solution of iron alum is suggested as a convenient method for reducing the intensity of the silver image in sections, when that is desired. Replacing the silver image with gold allows it to survive more drastic subsequent treatment, such as periodic acid oxidation.