Effect of temperature on argyrophil impregnation: development of a high temperature rapid argyrophil procedure

Our studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

A New Method for Demonstrating Argyrophil Cells of the Pancreas and Intestines

A new method for demonstrating argyrophil cells of the pancreas and intestinal tract using a combined silver and reducing solution in sections of formaldehyde fixed tissue is described. Impregnating sections in a 60 C water bath, the procedure takes about 25 min. A microwave version that takes about 5 min is also given. Results are similar to those obtained with the Grimelius method for argyrophil cells.     

A new method for demonstrating argyrophil cells of the pancreas and intestines

A new method for demonstrating argyrophil cells of the pancreas and intestinal tract using a combined silver and reducing solution in sections of formaldehyde fixed tissue is described. Impregnating sections in a 60 C water bath, the procedure takes about 25 min. A microwave version that takes about 5 min is also given. Results are similar to those obtained with the Grimelius method for argyrophil cells.     

A New Method for Easy Demonstration of Argyrophil Cells

A new method for silver impregnation of endocrine cells of the gastrointestinal mucosa is described. It offers great reliability, eveness of impregnation, and, since it can be used on batches of slides, is also suitable for histology class and investigation material. The procedure for paraffin sections of formalin-fixed material is as follows: dewax and transfer to distilled water, leave in 0.5% silver nitrate solution for 2 hours at 60 C. Rinse in distilled water, then treat in Bodian developer (hydroquinone, 1 g; sodium sulphite, 5 g; distilled water, 100 ml) previously heated to 60 C. Rinse in running tap water, distilled water, and then re-impregnate for 10 minutes at 60 C in the same silver solution and reduoc in Bodian's solution. Sma the background is not impregnated by this method, sections may be counterstained by any basic anilin dye to bring out nuclei. A 0.1% kernechtrot solution was found very satisfactory in this respect. The granulations of argyrophil cells stand out sharply black against a red background.     

Endocrine cells of the human gastric mucosa

Summary By light and electron microscopy investigation of the human gastric mucosa five types of ultrastructurally different endocrine cells have been detected: 5-hydroxytryptamine storing enterochromaffin (EC) cells, gastrin storing G cells, and functionally undefined ECL, D and D₁ cells. By direct application of Masson's argentaffin reaction as well as of Sevier-Munger's and Grimelius' argyrophil method to electron microscopy specimens, selective deposition of silver grains upon the endocrine granules of such cells was obtained. In particular, only EC cell granules reacted to the argentaffin method, granules of both EC and ECL cells heavily reacted to Sevier-Munger's technique, granules of EC, ECL, G and D₁ cells reacted to Grimelius' technique, while D cell granules failed to react either to argentaffin or argyrophil methods. By the application of the same silver methods to paraffin sections as well as by other selective staining methods for endocrine granules (5-hydroxytryptamine techniques, lead-haematoxylin, HCl-basic dye method), at least four of the above cell types were also identified under light microscope. This opens the way for extensive studies of such cells in conventional histologie specimens.This investigation was supported in part by grant N.70.01022.04 from the Italian Consiglio Nazionale delle Ricerche.     

Silver stains for identification of neuroendocrine cells. A study of the chemical background

Summary The chemical background of silver stains used for visualization and characterization of peripheral neuroendocrine cells in the gastrointestinal tract and pancreas, and of their corresponding tumours, was studied in tissue sections and by a dot-blot technique. Sequential staining of pancreatic islets with an immunohistochemical procedure and silver staining of the same tissue section revealed that chromogranin A immunostained cells also displayed an argyrophil reaction with the Grimelius method, but no argentaffin reaction with the Masson technique. Accordingly, purified chromogranin A (15 g or less) treated in formalin and applied to nitrocellulose did not show any argentaffin reaction but displayed a dose-related argyrophil reaction. Equal quantities of other polypeptide components did not give rise to any silver reaction. Further dot-blot studies showed that the tryptophan and tyrosine metabolites, dopamine, norepinephrine, 5-hydroxytryptamine and 5-hydroxindole caused strongly argentaffin and argyrophil reactions while epinephrine, 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophan gave only the former reaction. Among other chemical components studied, only guanine displayed weak silver staining. The results indicate that the reaction products between aldehydes and the granular content of biogenic amines synthesized from tryptophan and tryosine display an argentaffin reaction and that the granular chromogranin A caused an argyrophil but no argentaffin reaction.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

多疣壁虎肠道内分泌细胞的分布及形态学观察

用光镜和电镜对多疣壁虎肠道嗜银细胞的分布及形态做了初步的观察,结果显示,十二指肠嗜银细胞密度最高,大肠次之,空、回肠较低;作者认为嗜银细胞的分布与动物生活环境是相关的,嗜银细胞形态多样,位于肠上皮细胞之间和固有层结缔组织中,电镜下,嗜银细胞内充满足电子致密颗粒,十二指肠和大肠嗜银细胞颗粒大小和密度不同。根据内分泌特点的不同,可分为开放型和闭合型两类。本文还对多疣壁虎肠道嗜银细胞的分布及形态特点做了探讨。     

Neuron-specific enolase in mucosal endocrine cells and carcinoid tumours of the small intestine: A comparative study with neuron-specific enolase immunocytochemistry and silver stains

Summary Endocrine cells of human small intestinal mucosa, small intestinal carcinoids and carcinoid liver metastases were stained with an immunocytochemical technique using an antiserum against neuron-specific enolase (NSE), with the argyrophil technique of Grimelius and with the argentaffin technique of Masson. In the normal mucosa, scattered NSE-immunoreactive cells were seen mainly in the deeper parts of the crypts. These cells, as shown in the same sections, corresponded to the argentaffin and/or argyrophil cells indicating that they were of endocrine type.All intestinal carcinoids (16 cases) displayed NSE immunoreactivity. However, this reaction did not correlate on the cellular level with the silver techniques employed. Thus, many tumour cells were NSE immunoreactive but lacked an argentaffin or argyrophil reaction and vice versa. On the light microscopical level the silver techniques reveal the presence of neurohormonal granules in the tumour cells, while the NSE immunoreactivity appears to disclose neuroendocrine differentiation of the tumour cells irrespective of their hormone and granular content.Out of 13 carcinoid liver metastases, eight displayed strong NSE immunoreactivity, three were weakly stained and two were unreactive. Consecutive or the same tumour sections showed an argentaffin and argyrophil reaction in all carcinoid metastases. Since silver staining provides one type of information and NSE immunocytochemistry another, they provide in combination a good discriminator for neuroendocrine tumours.     

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.     

Electron microscopic identification of the histamine-storing argyrophil (enterochromaffin-like) cells in the rat stomach

Summary In the oxyntic gland area of the rat stomach the histamine-containing epithelial cells (also referred to as enterochromaffin-like cells because of their morphologic similarity with the 5-hydroxytryptamine-storing enterochromaffin cells) constitute the system of argyrophil cells in this area as previously shown by the combined use of fluorescence and light microscopic techniques. By performing the argyrophil staining reaction directly on ultra-thin sections it could be demonstrated in the electron microscope that the argyrophil cells have features suggesting that they are endocrine. Based on the ultrastructure of their secretory granules at least two such endocrine cell systems—both argyrophil—could be recognized in the oxyntic glands. The silver deposits were accumulated over the secretory granules of both these cell systems.It is well known that after injection of 1-3,4-dihydroxyphenylalanine, the histamine-storing (enterochromaffin-like) cells of the oxyntic glands store also dopamine. Under these conditions the enterochromaffin-like cells stain argentaffin, which has been shown at the light microscopic level. Also this reaction could be performed directly on ultra-thin sections. By electron microscopy it was then established that the two endocrine cell systems of the oxyntic gland area stained argentaffin upon treatment with 1-3,4-dihydroxyphenylalanine, and that the staining was confined to the secretory granules.The results clearly show that the enterochromaffin-like cells of the rat oxyntic gland area (which is devoid of 5-hydroxytryptamine-containing enterochromaffin cells) are identical with cells characterized as endocrine by ultrastructural criteria, and that gastric non-mast-cell histamine occurs in at least two separate systems of enterochromaffin-like cells.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.     

白条草蜥消化道嗜银细胞的分布和形态学观察

应用Grimelius银染法对白条草蜥消化道嗜银细胞进行了染色和切片观察.结果 表明:在白条草蜥的消化道中嗜银细胞分布广泛,见于其全长.其嗜银细胞形态多样,主要有锥体形、椭圆形等;其嗜银细胞分布密度于胃幽门最高,胃体次之,十二指肠最低.其消化道嗜银细胞分布型的形成与生活环境和取食方式等有关;根据其形态,嗜银细胞具有内、外分泌两种功能.     

Histochemical features of the Muscovy duck small intestine during development

We demonstrated for the first time the distribution and morphology of argyrophil and of goblet cells in the mucosa of the small intestine of the Muscovy duck during development using the Grimelius silver staining and alcian blue/periodic acid-Schiff (AB/PAS) staining technique. The argyrophil cells distribution was variable over the length of the small intestine from embryonic day 24 (24E) to post-hatching day 13 (13d). In the villi most argyrophil cells belonged to the open-type, while in the crypts they belonged to the closed-type. In the duodenum the density of argyrophil cells was highest at hatching, while in the jejunum and in the ileum the highest density value was at hatching and 13d. AB/PAS-positive goblet cells appeared on the villi and crypts of the duodenum and jejunum at 30E, and in the ileum at hatching. The density of AB/PAS-positive cells was the highest in the three segments at hatching. The AB-positive cells, compared with the PAS-positive cells, predominated in villi and crypts of the three segments, moreover the rate of AB-positive cells to PAS-positive cells significantly decreased from 30E to 9d. An increase in argyrophil and goblet cells number during the later incubation and at hatching, could indicate the small intestine in that period is being prepared to face a new diet.     

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections

Summary The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.     

凹耳蛙消化道组织学和嗜银细胞形态观察

为了揭示凹耳蛙(Odorrana tormota)消化道的基本特征,运用石蜡切片法和龙桂开银浸法对凹耳蛙消化道组织学结构及嗜银细胞的形态与分布密度进行了观察。结果显示:①凹耳蛙的胃壁具明显的纵行皱襞和胃小凹,胃腺发达,小肠可分为十二指肠和回肠,杯状细胞分散在十二指肠上皮细胞之间,十二指肠中未见十二指肠腺分布。②凹耳蛙嗜银细胞见于消化道全长,呈毛笔头样、锥体形、梭形、椭圆形和长条形等;幽门腺上皮和十二指肠绒毛上皮中的嗜银细胞具指向腺泡腔或肠腔的突起,提示其可能具有腔分泌的功能。嗜银细胞的分布密度胃幽门部最高,十二指肠和胃体其次,食道最低。据此认为胃既是凹耳蛙的主要消化器官,也是消化道中主要的内分泌器官;十二指肠是凹耳蛙消化道中的主要吸收部位,同时也具有内分泌功能;消化道嗜银细胞具有内分泌的功能,还可能具有腔分泌的功能。     

Fast and sensitive silver staining of DNA in polyacrylamide gels.

The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).