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.     

Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner

The periodic acid-methenamine silver staining technique, which is frequently used for demonstrating the renal glomerular basement membrane, requires a high degree of skill, and in some cases it may be difficult to obtain a good result. To overcome such difficulty and inconsistency, we have improved the method by performing methenamine silver staining after oxidation with periodic acid and subsequent application of thiosemicarbazide. In this procedure, this semicarbazide enhanced the reaction of methenamine silver with the glomerular basement membrane and the reaction was completed within a shorter time in comparison with the conventional method. This modification also eliminated any nonspecific reaction with the surface of the glass slide and the solution container and yielded excellent and reproducible results irrespective of the fixation method and material employed. It was also found to stain the renal glomerular basement membrane of rabbits, which is demonstrable only with difficulty by the conventional method.     

Thiosemicarbazide Used After Periodic Acid Makes Methenamine Silver Staining of Renal Glomerular Basement Membranes Faster and Cleaner

The periodic acid-methenamine silver staining technique, which is frequently used for demonstrating the renal glomerular basement membrane, requires a high degree of skill, and in some cases it may be difficult to obtain a good result. To overcome such difficulty and inconsistency, we have improved the method by performing methenamine silver staining after oxidation with periodic acid and subsequent application of thiosemicarbazide. In this procedure, this semicarbazide enhanced the reaction of methenamine silver with the glomerular basement membrane and the reaction was completed within a shorter time in comparison with the conventional method. This modification also eliminated any nonspecific reaction with the surface of the glass slide and the solution container and yielded excellent and reproducible results irrespective of the fixation method and material employed. It was also. found to stain the renal glomerular basement membrane of rabbits, which is demonstrable only with difficulty by the conventional method.     

Alterations in the distribution of glycoproteins in epithelial cells of murine colon after injection of tunicamycin

Summary Glycoproteins are associated with several structures of colonic absorptive cells of the mouse. These include the cell coat, Golgi apparatus and vesicles that transport the glycoproteins from the apparatus to the cell surface (Michaels and Leblond 1976). In many in vitro systems, the antibiotic tunicamycin inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins. In the present in vivo study, tunicamycin was injected into mice. The murine colonic epithelial cells were prepared routinely for electron microscopy and cytochemistry. Cells from the experimental and control animals were similar morphologically. However, staining by the periodic acid-chromic acid-silver methenamine technique, revealed differences in the distribution of glycoproteins. In animals that received the higher dosages of tunicamycin there was a substantial reduction in silver staining in both the Golgi apparatus and the vesicles of colonic epithelial cells compared to these structures in cells of identically treated control tissues, whereas the staining over the cell coat was not significantly altered. Possible explanations for the staining of the cell coat in the treated animals were provided in the text. This report demonstrates the feasibility of using tunicamycin in vivo and detection of the changes obtained by the silver methenamine method.     

Silver methenamine and phosphotungstic acid staining of the acrosome of Mytilus edulis.

The Mytilus acrosome was investigated by histochemical methods combined with electron microscopy, using silver methenamine (SM) and phosphotungstic acid (PTA) staining, as well as some chemical and enzymatic pretreatments followed by the staining. As one of two major components in the Mytilus acrosome, the egg-membrane lysin was conspicuously stainable with PTA and susceptible to pronase digestion. The other component, that occupies the space between the acrosomal membrane and the axially located strand containing lysin, was stained with SM very specifically. This staining property was not affected by pronase digestion or treatment that blocked aldehyde and SH groups.     

Demonstration of the polysaccharides in the cell wall of Candida albicans blastospores,using silver methenamine staining and a sequence of extraction procedures

Blastospores of Candida albicans were subjected to a series of extractions of increasing severity to remove polysaccharides from the wall. The procedure was observed by electron microscopy, using silver methenamine staining to localize the polysaccharides.     

Histochemical staining of the complex carbohydrates of the midgut of the mosquito,Culex tarsalis coquillett

Histochemical staining of the midgut epithelial cell surface complex carbohydrates of the mosquito Culex tarsalis was examined electron microscopically. The microvillar surface is composed primarily of neutral vic-glycoconjugates; positively stained by silver methenamine and silver protein. Lanthanum and alcian blue staining indicate that the microvilli contain a minimal anionic component; possibly phosphoglycoconjugates. Similarly, the intercellular junctions contain a predominance of neutral vic-glycoconjugates. In addition, the intercellular junctions contain fixed positive charges, based on en bloc phosphotungstic acid staining. The midgut basolateral membrane system and the basal lamina are both highly anionic; stained by ruthenium red, tannic acid, alcian blue and periodic acid-chromic acid-phosphotungstic acid. The basolateral plasma membrane also contains some vic-glycoconjugates. Selective staining indicates that the anionic component of the basolateral plasma membrane and the basal lamina is predominantly carboxyl groups; no specific staining for sulfo- or phosphoglycoconjugates was observed.     

A combined fluorescent and electron microscopic stain for elastic tissue

Synopsis A histochemical procedure for fluorescent and electron microscopic observations of elastic tissue has been developed. This was accomplished by combining thein vivo andin vitro staining properties of non-metallic tetraphenylporphine sulphonate and its silver derivative. The non-metallic porphyrin gives a bright red fluorescence to elastica after injection, but it does not make the tissue electron dense. However, the metallic derivative imparts a specific electron density to elastica afterin vitro staining, but it does not render elastica either fluorescent or electron dense after injection. Thus, elastic tissue can be viewed with the fluorescence microscope after injection of the nonmetallic porphyrin. Sections from the same block can be stainedin vitro with silver tetraphenylporphine sulphonate and observed in the electron microscope. The nonmetallic porphyrin readily crosses the placental barrier and, therefore, can be used to study the elastica in foetuses by fluorescence microscopy.     

Chemical and Spectrometric Evaluation of Glycogen After Routine Histological Fixatives

A detailed comparison of fixatives used for the demonstration of glycogen has been based on chemical assay and microspectrophotometry. Rat liver containing known amounts of glycogen was fixed in formol alcohol, Rossman's fluid, 10% neutral formalin, Bouin, Helly, SUSA, and Zenker's fluid at 4 C and 18 C. Chemical assay was carried out before and after fixation and paraffin sections were prepared from the fixed material. Sections were stained with PAS and the silver methenamine method. Visual examination was carried out with a comparison microscope and quantitative estimations on PAS-stained sections were performed by scanning microspectrophotometry. The histochemical methods were compared with the chemical results obtained from the same tissue and a reasonable degree of correlation between the sets of results was observed. Cold formol alcohol and cold Rossman's fluid preserved the most glycogen and Zenker and SUSA fixation preserved the least. Cold formol alcohol was the only fixative that preserved threshold values of glycogen i.e. 0.3% and the silver methenamine method is recommended for the demonstration of these small amounts.     

人羊膜与人脱细胞羊膜形态结构的对比观察

目的：对比观察人羊膜（human amniotic membrane,HAM）脱细胞处理前后的形态结构变化,为人脱细胞羊膜（human acellu-lar amniotic membrane,HAAM）作为良好的生物支架材料提供依据。方法：取健康剖宫产孕妇的胎盘,剥离获取HAM,行HE染色、透射电镜（transmission electron microscope,TEM）、上皮面与基质面扫描电镜（scanning electron microscope,SEM）检测;将HAM经物理和胰蛋白酶等脱细胞处理后获得HAAM,亦行HE染色、TEM、上皮面与基质面SEM检测;最后将检测结果进行对比观察。结果：通过HE染色表明HAM的细胞成分去除干净;TEM断面观察HAAM表明其富含大量密集的呈点状、线状及条索状的纤维成分;SEM观察表明HAAM的上皮面与基质面呈现不同的三维结构,未见胶原纤维和网状纤维断裂。结论：HAM经脱细胞处理制备的HAAM,既去除了可引起移植排斥反应的细胞成分,又保存了完整的三维结构,为良好的生物支架材料。     

Location of Polysaccharide on Chlamydia psittaci by Silver-Methenamine Staining and Electron Microscopy

Previous serological studies have indicated that the group antigen of chlamydial organisms is composed of an acidic polysaccharide and a lipid component. The present study was undertaken in an effort to locate this polysaccharide complex by use of electron microscopy and a silver-methenamine marker. The meningopneumonitis strain of Chlamydia psittaci was propagated in HeLa-M cell culture. Organisms were purified by differential centrifugation, treatment with Genetron, and by gel filtration. After fixation and embedding, sections were obtained for electron microscopy. Sections were stained for carbohydrates with silver-methenamine. A double layer of regularly spaced silver grains of uniform size was observed at the periphery of the sectioned organisms tracing the contours of the surface membrane (cell wall). This intensity of staining was observed only when sections were oxidized with periodate prior to silver-methenamine staining. Prior treatment with 1% sodium deoxycholate resulted in a significant reduction in staining. It is considered probable that the periodate-sensitive polysaccharide found at the periphery of the organisms represents, or is a component of, the group antigen of these organisms.     

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.     

用DNA银染法研究传染性软疣病毒的形态发生发育过程

用ＤＮＡ银染技术显示了传染性软疣病毒（ＭＣＶ）形态发生发育及其过程中ＤＮＡ的变化。结果显示：在被感染的皮肤表皮细胞内都有一个大小及构型不同的银染区（病毒工厂）。ＭＣＶ的发生发育均在银染区内而不在胞质区内。其发生过程是先在细胞一侧的胞质内复制合成病毒ＤＮＡ等物质,形成中等密度的银粒大小不等的银染区（病毒前包涵体区）,然后在其中形成致密的细粒状银染区（病毒前基质区）,接着在后者的周围出现弧形的粗粒银沉淀（初期ＭＣＶ的外膜）,逐渐分割包围病毒前基质而形成初期ＭＣＶ。在发育过程中,初期ＭＣＶ的外膜、基质,核心外膜及核心均经过一系列的形态变化。侧体是中期ＭＣＶ向成熟期发育中出现的暂时性结构,其本质为含ＤＮＡ成分的病毒基质。本研究提示：ＭＣＶ的ＤＮＡ物质进入皮肤表皮细胞后能大量复制,合成大量的病毒蛋白质,自主地装配成完整的初期ＭＣＶ,后者有独特的形态发育过程。     

Ultrastructural localization of carbohydrates on thin sections of Staphylococcus aureus with silver methenamine and wheat germ agglutinin-gold complex.

Postembedding staining of intracellular carbohydrates on thin sections of Staphylococcus aureus was studied by the silver methenamine and the wheat germ agglutinin-gold techniques. Staining of silver grains was observed on both the cell wall and the cross wall. The staining was interpreted to be due to teichoic acid. Labeling by wheat germ agglutinin-gold particles was observed on both the cell wall and the cross wall, and the staining pattern resembled that of silver methenamine staining. Therefore, the labeling was considered to be due to N-acetylglucosamine of teichoic acid. The combination of two types of cytochemical techniques was useful to localize and characterize the carbohydrates of the bacterial cell.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

胎盘脱细胞基质作为组织修复材料的初步研究

目的 制备胎盘脱细胞基质并评价其生物相容性，探讨其作为组织修复材料的可行性。方法 利用分娩废弃物胎盘组织，进行病毒灭活、脱细胞处理、冷冻干燥得到胎盘脱细胞海绵状基质材料，HE染色观察脱细胞效果及扫描电镜观察材料微观结构。同时，选取健康雄性SD大鼠39只，体质量120～150 g，随机分为实验1组、实验2组及对照组。对构建的基质材料进行大鼠皮下植入实验，实验1组植入基质材料，实验2组植入基质材料及脐带间充质干细胞，对照组为假手术组。于手术后第3、5、7天进行动物血常规检测，分析淋巴细胞、粒细胞等炎性细胞数量；于手术后第1、2、4、8、9周取材料植入处及周围组织样本进行HE染色分析。结果 构建的胎盘脱细胞基质肉眼观呈乳白色海绵状，HE染色未见细胞残留，电镜观察材料内部空隙比较明显，材料交联度较好，总孔隙率为（77.54±2.53）%。皮下植入之后，切口处愈合良好，血常规结果未见明显的炎性细胞增多；术后7 d植入材料切片HE染色即可见血管形成，且脐带间充质干细胞的加入能够加快材料与机体的融合，促进细胞的深入生长及血管化。结论 胎盘脱细胞海绵基质材料具有良好的生物相容性，可作为组织工程材料的理想来源。     

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.     

HISTOCHEMICAL STAINING AND CHARACTERIZATION OF GLYCOPROTEINS IN ACID-SECRETING CELLS OF FROG STOMACH

Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.     

Mucosubstances in the normal human oesophageal epithelium

Summary Normal human oesophageal epithelium was investigated with the periodic acid — silver methenamine technique and its variations to demonstrate neutral mucosubstances at the ultrastructural level. The results were compared with the acid phosphotungstic acid method. Neutral mucosubstances were shown in the cell coat and membrane coating granules by both techniques. The silver methods also demonstrated glycogen, the Golgi apparatus and dense bodies. The periodic acid — silver methenamine technique outlined positive material in the intercellular space of the prickle cell layer, but the other silver methods did not.     

Mucosubstances in the normal human oesophageal epithelium. A comparison of periodic acid-silver and phophotungstic acid techniques.

Normal human oesophageal epithelium was investigated with the periodic-acid-silver methenamine technique and its variations to demonstrate neutral mucosubstances at the ultrastruct level. The results were compared with the acid phosphotungstic acid method. Neutral mucosubstances were shown in the cell coat and membrane coating granules by both techniques. The silver methods also demonstrated glycogen, the Golgi apparatus and dense bodies. The periodic acid-silver methenamine technique outlined positive material in the intercellular space of the prickle cell layer, but the other silver methods did not.