Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.     

Electron dense artefactual deposits in tissue sections: the role of ethanol, uranyl acetate and phosphate buffer.

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

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     

Gilbert 综合征肝活检的超微结构特征*

目的:确定Gilbert综合征患者肝组织的超微结构特征,为Gilbert综合征的诊断和鉴别诊断提供新的方法。方法:按电镜常规进行标本制备,应用透射电镜对20例Gilbert综合征患者肝穿刺活检组织进行超微结构观察。结果:肝细胞可出现巨大线粒体,常含有副晶格样包涵体、较明显的基质致密颗粒。肝细胞常见脂褐素颗粒增多,多分布于毛细胆管周围肝细胞内。可出现较有特征性的色素颗粒,大小不等,卵圆形或不规则形,含有电子致密块状颗粒,与电子密度略低的聚集物以及脂滴互相混杂。这些溶酶体颗粒的基质由细小的、弱嗜锇性的颗粒组成。少数颗粒类似Dubin-Johnson综合征的颗粒。但颗粒较小,缺少致密核芯结构。结论:特征性的含粗大电子致密物的溶酶体对Gilbert综合征的诊断有重要参考价值。     

Methods for removing uranyl acetate precipitate from ultrathin sections.

Precipitate resulting from en bloc staining with uranyl acetate was removed by treating sections with 15% oxalic acid in 50% methanol for 30 minutes at 40 C. Precipitate resulting from poststaining sections with hot uranyl acetate was removed by rinsing sections in 0.25-0.50% aqueous oxalic acid for 10-15 seconds at room temperature. Rinsing sections for longer than 30 seconds removed uranyl precipitate and also destained the sections. These procedures did not damage the embedding medium or cellular detail.     

Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland.

Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.     

Identification of pancreatic glucagon cells in the cartilaginous fish Scyliorhinus canicula by ultrastructural immunocytochemistry

The ultrastructural localization of glucagon in the presence of Scyliorhinus canicula was investigated. We used a post-embedding immunoelectron microscopy method on pancreatic samples fixed in glutaraldehyde and osmicated before embedding. Contrasting with uranyl acetate and lead citrate was also performed after immunolabelling, but best results were obtained with uranyl acetate only. Glucagon-like immunoreactivity was located in round granules (300-600 nm) surrounded by a limiting membrane. The matrix varied in electron density and exhibited a dense core surrounded by a less dense mantle. The granules were seen in two different cell types, which differed in the electron density of their cytoplasm. Glucagon-immunoreactive cells were the largest pancreatic cells types and were often localized near somatostatin-containing cells.     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland

Summary Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.     

Alkaline bismuth solution as an en bloc stain for formaldehyde-glutaraldehyde potassium permanganate fixed fungal spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.     

Identification of helical polyribosomes in sections of mature skeletal muscle fibers

Summary The localization and configurations of ribosomes in mature white skeletal muscle fibers of the rat were investigated. Differential visualization of ribosomes and glycogen granules was obtained by fixation with glutaraldehyde only and staining of the sections with uranyl acetate. Ribosomes are then electron dense and glycogen granules electron transparent. Their identity was ascertained by selective extractions of ribonucleic acid and polysaccharide.The vast majority of the ribosomes is not membrane-bound. They are located intermyofibrillarly (predominantly at the level of the I-bands), beneath the sarcolemma, and in the paranuclear cones of sarcoplasm. Occasionally short stretches of granular reticulum occur, often as characteristic double walled vesicles with ribosomes on the inner membrane only.Three main types of polysomal configurations are observed: rosettes of 4 to 6 ribosomes, helical arrays, and whorls of up to about 25 probably membrane-bound ribosomes. The average number of ribosomes in the extended helical configurations is estimated to be about 60. It is argued that these helices represent the polysomes instrumental in the synthesis of the large subunits of myosin. It is emphasized that helical polyribosomes are by no means typical of striated muscle, but probably represent a common configuration of large free polysomes.With the technical assistance of Tineke J. Hoogenboezem.     

Extraction of osmium-containing lipids by section staining for TEM

Postfixation with osmium-ferrocyanide or OSO4 renders lipid droplets in rat liver and kidney homogeneously electron dense without additional section staining. In sections of the same block that have been single stained by uranyl acetate or lead citrate, lipid droplets show a more electron translucent center surrounded by a dense rim. In sections double stained with uranyl acetate and lead citrate, lipid droplets frequently appear as empty vacuoles, from which the electron dense content has been completely extracted.     

Block-staining tissues with potassium ferrocyanide-reduced osmium tetroxide and lead aspartate for electron microscopic radioautography

In the hope of devising a method for prestaining tissues en bloc for electron microscopic radioautography, pieces of radioiodine-labeled liver were taken through various combinations of ferrocyanide-reduced osmium tetroxide, lead aspartate, and aqueous uranyl acetate at room temperature or at 60 degrees C. Following the tests, the method adopted for routine use was to block-stain tissues for 2 hr in potassium ferrocyanide-reduced osmium tetroxide at 4 degrees C followed by 1 hr in Walton's lead aspartate at room temperature. This simple method, which requires no manipulation before or after emulsion coating and development of the radioautographs, provides adequate contrast without inducing background fog or artifacts.     

Size changes in single muscle fibers during fixation and embedding.

During fixation of single muscles fibers with glutaraldehyde, the volume of the fiber shrinks 20%, recovers in rinse and osmium tetroxide to near normal volume and shrinks 20% again when staining with uranyl acetate. This suggest that osmotic properties of membranes may not have been completely lost during fixation, post-fixation and en bloc staining. Dehydration in ethanol and propylene oxide produces a further 10% shrinkage in volume. Infiltration and embedding with Epon causes an additional 15% change in volume. This gives a total shrinkage in volume of 45% which is nearly twice that of the apparent shrinkage in the volume of the myosin lattice as determined by electron microscopy.     

Electron Dense Artefactual Deposits in Tissue Sections: The Role of Ethanol, Uranyl Acetate and Phosphate Buffer

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

Isolation of liver lysosomes by iron loading. Ultrastructural characterization

In summary, the data demonstrate that, by the use of repeated injections of an iron sorbitol complex, it is possible to isolate a fraction highly enriched in hydrolytic enzymes (60 times over the homogenate) and in well preserved lysosomes emanating almost entirely from liver parenchymal cells. The advantage of adding fixative to the bottom of the gradient and of using en bloc staining with uranyl acetate is also demonstrated.     

Collagen formation — observations on its intracellular packaging and transport

Summary Odontoblasts, osteoblasts and fibroblasts of young rats were examined in the electron microscope after staining thin sections either with lead citrate alone or with uranyl acetate prior to lead citrate.With lead citrate alone, collagen fibrils in the extracellular matrix stand out as lucent structures against a moderately electron dense background. Within the cells, lucency is restricted to certain dilated portions of the Golgi saccules as well as to the secretory granules located nearby and in the secretory pole of the cells. The lucency present in these compartments may be attributed to fibrils that are similar to the lucent collagen fibrils in the extracellular matrix. Other cellular compartments, e.g. the rough ER, do not display lucency.When preparations are stained with uranyl acetate prior to lead citrate, lucency is observed neither in the matrix nor in the cells. In the matrix, collagen fibrils are easily identifiable by their cross banded pattern. In the odontoblasts, dilated portions of Golgi saccules between the outer and inner face contain filaments aligned in parallel that are approximately 3 000 Å in length. In saccules on the inner face filament aggregates are present, some of them exhibiting a cross banding pattern. In secretory granules, however, the contents appear rather homogeneous.It is suggested that filament aggregates of collagen can assemble in the Golgi apparatus from filamentous units. These are transported through the cell by way of secretion granules and are discharged to the extracellular matrix by exocytosis.This investigation was supported by grants of the Medical Research Council of Canada. The author wishes to express appreciation to Dr. C. P. Leblond for his guidance in the course of this work.     

Masking of pleomorphic glycogen sites by methanolic uranyl acetate.

Rat liver tissue was fixed in 2.5% glutaraldehyde buffered with cacodylic acid (pH 7.3) for 2 hr, washed twice in buffer, and postfixed in 2% osmium tetroxide at 4 C for 1 hr. The tissue then was dehydrated, infiltrated with and embedded in Epon by routine procedures. The ultrathin sections from this tissue, when stained with spectroscopic grade methanol saturated with uranyl acetate (SMUA) for 1 min followed by aqueous lead citrate (PbCi) (Reynolds 1963) for 5 min at room temperature, showed a uniform staining of all major cellular components except glycogen. The SMUA appeared to be specific for ribonuceloprotein granules, rendering them more prominent in the cytoplasm due to the lack of glycogen staining. The question of glycogen removal from the sections due to SMUA treatment was evulated using various extractions and staining methods. It appeared that SMUA pretreatment alters the subsequent binding ability of lead salts, resulting in lack of glycogen staining, although it does not remove the glycogen from the sections.     

A simple method of staining juxtaglomerular granules in the rat kidney for high resolution light microscopy

A simple method for the demonstration of juxtaglomerular granules in Epon embedded semithin (0.5-1 micrometer) sections has been developed as follows: sections are prepared as for routine electron microscopy except that before dehydration, the tissues are immersed in 0.5% uranyl acetate in Veronal acetate buffer (pH 5.0) overnight at room temperature. After sectioning on an ultramicrotome, the semithin sections are briefly stained with toluidine blue-pyronin Y. After staining, the section is rinsed in running tap water and then air dried. Under a light microscope with a 40 X or a 100 X objective, the juxtaglomerular granules appear as deep purple particles and are thus easily separated from the bluish cytoplasm of the juxtaglomerular cells. Cellular organelles in other cells of the kidney were also clearly stained and their fine structure distinguishable.