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.     

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.     

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     

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.     

小鼠精母细胞联会复合体RNA组分的电镜研究

本文运用常规染色和Bernhard染色方法对切片标本中小鼠粗线期精母细胞联会复合体(SC)的超微结构和电镜细胞化学特点进行了研究。经常规染色后,可见SC由侧生组分(LE)、中央组分(CE)和L-C纤维组成;SC宽约210nm,LE宽约60nm,中央间隔区宽约90nm。在Bernhard染色标本中,SC的LE、CE和L-C纤维着色较深,说明其中含有RNA;SC各结构组分的宽度和形态特点与常规染色标本中的基本一致。本文讨论了SC中存在有RNA等问题。     

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.     

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 proocdures. The ultra thin 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 allular components ercept glycogen. The SMUA appeared to be specific for ribonucleoprotein 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 evaluated 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.     

Lipid droplets in the adrenal cortex of the rat. Preservation after tannic acid—paraformaldehyde—glutaraldehyde fixation and extraction during staining

Adrenocortical tissue from the rat was fixed in glutaraldehyde-paraformaldehyde-tannic acid with or without potassium pyroantimonate. An electron opacity was observed in lipid droplets from unstained sections of tissue with or without antimonate in the fixative and is most likely attributable to inclusion of tannic acid in the fixative. The opacity was largely removed after staining with uranyl acetate in absolute methanol followed by lead citrate. Removal of the opacity is attributable to staining in lead citrate, not uranyl acetate, because highly basic solution without lead also removes the density. An electron-opaque rim is present at the interface of lipid droplet and cytoplasm, although no distinct membranous structure is observable. The rim may correspond to myelin-like structures seen sometimes in lipid droplets from adrenocortical cells fixed by routine procedures employing pre-fixation with glutaraldehyde and post-fixation with osmium tetroxide. Results of this study point to the conclusion that ultrathin sections should be examined unstained in the validation of a new regime for processing tissues in electron microscopy.     

Ruthenium Red Stain Able Surface Layer on Lung Alveolar Cells; Electron Microscopic Interpretation

Luft's ruthenium red (RR) method was applied to lung tissue. Small blocks of mouse lung were fixed for 1 hr with 1.2% glutaraldehyde at 0-4 C, buffered with 0.067 M cacodylate, pH 7.3 and containing RR, 1 mg/ml. Following fixation, lung blocks were immersed in 0.15 M cacodylate for 10 min and postfixed for 3 hr at room temperature with 2% OsO₄ buffered with 0.067 M cacodylate, pH 7.3, and containing RR, 1 mg/ml. Blocks were dehydrated with ethanol, embedded in Araldite, and ultrathin sections treated with uranyl acetate and lead citrate solutions to enhance contrast of cell structures. Electron micrographs revealed an electron-dense layer coating the exposed surfaces of alveolar cells. This layer corresponded in location and appearance to that observed by other investigators who used colloidal iron techniques.     

PREFERENTIAL STAINING OF NUCLEIC ACID-CONTAINING STRUCTURES FOR ELECTRON MICROSCOPY

Oriented fibres of extracted nucleohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tetroxide solution at pH 6, containing 10^-2 M Ca⁺⁺, and embedding in Araldite enabled sections of the fibres to be cut in which the orientation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqueous solution. Strongest staining of whole tissue was obtained with very short fixation times-5 minutes or so at 0°C. Particularly intense staining was obtained when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissues fixed for longer times (½ hour to 2 hours, at 0°C or 20°C), in briefly fixed material the DNA-containing regions appeared relatively unstained by lead hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.     

Kinetics of Lead Citrate Staining of Thin Sections for Electron Microscopy

The staining of thin sections with lead citrate shows an initial increase followed by a decrease much later; the rate of the initial increase and subsequent loss varies for different cellular components. The decrease eventually reaches a stable minimum. At this level electron scattering is less than that of unstained sections, demonstrating a loss of biological material.

Lead citrate used as a poststain following uranyl acetate causes an increase in electron density that is independent of staining time over 1-30 rain; this increase appears to depend only on the quantity of uranyl acetate already bound, implying that the lead binds predominantly to the uranyl acetate.     

The loss of enzyme reaction products from ultrathin sections during the staining for electron microscopy

Summary The effects of heavy metal salt staining procedures on the reaction products obtained in the demonstration of arylsulphatase and of acid phosphatase were studied.Lead citrate staining at pH 12 was found to cause a very marked dissolution of barium sulphate and a moderate dissolution of lead sulphate. The staining with uranyl acetate was found to dissolve moderately both barium and lead sulphate.Neither lead citrate nor uranyl acetate staining had any remarkable effect on lead phosphate.The mechanism of the dissolution and the possibilities to avoid it were discussed.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of the pond snail, Cipangopaludina malleata Reeve, were fixed in 1% osmium tetroxide, 3% permanganate, or 4% formaldehyde followed by 1% osmium tetroxide, each being buffered to pH 7.2 with Veronal-acetate or Sörensen's phosphate buffer. On the other hand, testes fixed with 4% formaldehyde adjusted to pH 7.2 with 0.075 M Na-cacodylate were incubated in Novikoff-Goldfischer medium for demonstrating thiamine pyrophosphatase, uridine or inosine diphosphatase, uridine monophosphatase or adenosine triphosphatase. The specimens incubated were postfixed in 1% osmium tetroxide buffered to pH 7.2 with Veronal-acetate buffer. Thin sections of the epoxy Epon resin-embedded tissue were stained either singly with saturated aqueous uranyl acetate or doubly with saturated aqueous uranyl acetate followed by lead citrate.In a concentric lamellar structure consisting of the granular endoplasmic reticulum in the cytoplasm of early atypical spermatids, disappearance of ribosomes attached to the outer surface of cisternae seems to have initiated at the central part of the structure, and the cisterna-attached ribosomes seem to participate in the formation of dense granules appearing in the vesicles representing the endoplasmic reticulum of atypical spermatids.The Golgi apparatus of the atypical spermatids in the advanced stages of development is composed of at least three different layers, the central part consisting of an amorphous material, the following lamellar and vesicular elements, and the peripheral fine vesicles.It has been assumed that the mechanism by which the nucleic acid, especially DNA is converted into the polysaccharide might be attributed to the function of the Golgi apparatus, because the transformation of dense granules into less dense granules as well as diphosphatase activities have been detected within the Golgi apparatus.This study was supported by Grant GM-8327-06 from the United States Public Health Service.     

Ultrastructure of cardiac muscle of Trochosa terricola Thor., Pardosa amentata Clerck,P. pullata Clerck,and Pisaura mirabilis Clerck (Araneae: Lycosidae,Pisauridae)

Summary The ultrastructure of the cardiac muscle in some araneae has been investigated. The sarcolemma invaginates at the Z band and may extend into the cells through several myofibrils. Numerous T-tubules are given off both from sarcolemmal invaginations and also directly from the cell surface. In passing through the Z band, the luminal diameter of T-tubules greatly increases. Dyadic and a few triadic couplings are found mainly at the A-I level. Peripheral couplings were not seen.The ruthenium red solutions employed were prepared as described by Myklebust (1975), but the fixative contained 2% sucrose. The hearts were fixed for 3 1/2h in the ice cold solution, washed in buffer and post-fixed for 1 1/2h in a ruthenium red/OsO₄ solution. Dehydration and embedding was performed as described above. Sections were cut on a Reichert Om U2 microtome, stained with lead citrate (Reynolds, 1963) and examined in a Philips 300 electron microscope at 80 kV.Semithin sections for light microscopy were stained with toluidine blue (Mercer and Birbeck, 1972).I am much obliged to cand. real Erling Hauge for identifying the specimens used in the investigation proportion of relaxed sarcomeres. The fixed pieces were washed in a buffered sucrose solution and post-fixed in a 1% OsO₄ solution buffered with sodium cacodylate for 1 h. Following washes in a compatible buffer and in distilled water, the tissue was stained en bloc for l h in a 2% aqueous uranyl acetate solution. The tissue was dehydrated through cold acetone and embedded in Epon 812.     

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)     

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.     

Presence of glycoconjugates in prolactin granules of male rats

Summary Prolactin granules in the anterior pituitary glands of male rats contain densely stained materials at the periphery of the matrix. These occur in both small spherical and large polymorphic types of granules. The presence of densely stained materials around secretory granules may be a useful criterion for identification of prolactin cells since the dense structure was observed in 95% of these cells after conventional staining by uranyl acetate and lead citrate. The localization of glycoconjugates in the prolactin granules was examined by applying concanavalin A (Con A) on the ultrathin sections. HRP-Con A or ferritinconjugated Con A bound specifically to the densely stained materials in the peripheral region of the prolactin granule matrix, indicating that this densely stained matrix contains glycoconjugates; the significance thereof is discussed with reference to the concentration and packaging of secretory product.     

伦敦白胶和茶：一种既快又安全的用于透射电子显微镜的细菌样品制备方法

【目的】检测乌龙茶提取物是否可作为电子染色剂取代醋酸双氧铀用于细菌细胞染色,使其能在透射电子显微镜下进行观察。【方法】利用伦敦白胶对细菌样品(大肠杆菌和金黄色葡萄球菌)进行胶块的制备,再在复染铅与不复染铅这两种情况下对超薄切片样品进行3种不同染色剂的电子染色,之后在透射电子显微镜下观察比较其不同之处。这3种不同的染色剂分别是醋酸双氧铀、0.05%乌龙茶提取物以及0.1%乌龙茶提取物。首先将带有超薄切片样品的铜网悬浮于不同的待比较染液中10?15 min,若需进一步用柠檬酸铅复染,则将经3次蒸馏水冲洗过后的铜网再次悬浮于柠檬酸铅染液中8?10 min。【结果】复染铅的情况下,在透射电子显微镜下无论是大肠杆菌还是金黄色葡萄球菌,利用3种电子染色剂进行染色的结果均非常相似。【结论】实验结果表明,在观察细菌结构中,乌龙茶提取物可以替代醋酸双氧铀进行透射电子显微镜样品的电子染色。     

EVIDENCE FOR CHANGES IN PROTEIN POLYSACCHARIDE ASSOCIATED WITH THE ONSET OF CALCIFICATION IN CARTILAGE

Past work has suggested that protein polysaccharide may play a role in the calcification of cartilage. Recent electron microscopic studies on noncalcified cartilage have indicated that protein polysaccharide in cartilage matrix is represented by granules associated with collagen fibers. The present work has been designed for comparison of the matrix of noncalcified cartilage to that of calcified cartilage, with particular reference to these granules. Small blocks of tibia from 16-day embryos were fixed in cacodylate-buffered glutaraldehyde and postfixed in either phosphate- or Veronal-buffered osmium tetroxide. Special care was taken to maintain the pH above 7.0 at all times. For electron microscopy the tissues were dehydrated, embedded in Epon 812, sectioned, and stained with uranyl acetate or lead citrate. A marked decrease in the size of granules in the matrix of calcified cartilage compared to noncalcified cartilage was noted. Associated with the decrease in the size of granules was a condensation of matrix components and the presence of an amorphous electron-opaque material that was not seen in noncalcified areas. These results are interpreted to represent either a drop in concentration or a change in state of protein polysaccharide with the onset of calcification in cartilage.