Resolution of a gold latensification-elon ascorbic acid developer for Ilford L4 emulsion.

The electron-microscopical autoradiographical resolution of a gold latensification-elon ascorbic acid (GEA) developer for Ilford L4 emulsion was determined experimentally, using radioactive line sources of tritiated albumin (Heijnen and Geuze, 1977). For sections with a thickness of 62 nm (SD +/- 11), which were covered with a carbon layer about 5 nm thick and a slightly overlapping monolayer of L4 silver bromide crystals, the measured half-distance (HD) of resolution was 115 nm. This improvement in resolution, the high efficiency of the GEA developer for L4 emulsion (Wisse and Tates, 1968), and the excellent visibility of the cellular structures under the small silver grains, mean that the L4-GEA combination deserves preverence as a method for quantitative electron-microscopical autoradiography.     

Phenidone-ascorbic acid development in electronmicroscopic autoradiography

Summary Phenidone-ascorbic acid development was calibrated for electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold sections of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by crosssectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible.Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.     

Autometallography

Summary The autometallographic procedure represents a new technique that can substitute for the normal methods of physical development (PD). The physical developer (a solution of reducing substance, silver salt and protection colloid) is replaced by a photographic emulsion and chemical developer. Accumulations of gold, silver, metal sulphides and metal selenides can be amplified by the present technique.Tissue sections placed on glass slides are covered by a silver bromide containing emulsion, dried and exposed to a chemical developer. After development the emulsion is either removed or cleared and the sections are counterstained and embedded.The autometallographic procedure can also be applied to ultrathin sections.     

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters

Resolution for 125I-labeled specimens under electron microscope (EM) autoradiographic conditions was assessed experimentally. With this isotope the size of the silver halide crystal was the most important resolution-limiting factor. Heavy metal staining such as is routinely used in preparing animal tissues for EM autoradiography produced an improvement in resolution of approximately 15-20%. For a 500-1,000-A biological tissue section fixed with OsO4 and stained with uranyl acetate, we obtained resolution (half distance, HD) values of approximately 800 +/- 120 A using Ilford L4 emulsion and 500 +/- 70 A using a Kodak NTE-type emulsion. General aspects of resolution-limiting factors and comparison with 3H and 14C values are discussed.     

Assessment of resolution by half distance values for tritium and radioiodine in electron microscopic radioautographs using Ilford L4 emulsion developed by “Solution Physical” or D-19b methods

Summary Half distance values for electron microscopic (EM) radioautographs with the isotopes³H and¹²⁵I were determined using Ilford L4 emulsion processed with either fine grain, solution physical development, or filamentous grain, chemical development with D-19b.³H- and¹²⁵I-line sources, obtained by cutting perpendicular sections from sections of³H-labeled methacrylate or¹²⁵I-labeled thyroid glands, were processed for EM radioautography. The distribution of silver grains around a line source was determined by measuring their distance from the source in photographs of EM radioautographs. The number of silver grains per unit distance from the line source was plotted on graphs and half distance values were calculated. With solution physical development, the half distance value was 76 nm for³H and 80 nm for¹²⁵I; whereas with D-19 b development it was 187 nm for³H and 157 nm for¹²⁵I. Since solution physical development produced a reduction of about 50% in the half distance values for both isotopes, it is concluded that the production of fine grain by this method provides better resolution for EM radioautography than filamentous grain development with D-19 b. This work was the subject of a McGill University dissertation (Levine 1977)     

HIGH-RESOLUTION AUTORADIOGRAPHY : I. Methods

Methods used in obtaining high resolution in autoradiography, with special emphasis on the technique of electron microscopic autoradiography, are described, together with control experiments designed to establish the optimum conditions or procedures. On the basis of these experiments the emulsion selected was Ilford L-4, with a crystal size slightly larger than 0.1 micron. It is applied to the specimen in the form of a gelled film consisting of a monolayer of silver halide crystals. Background, when present, can be eradicated by a simple method. The preparations can be stored, in presence of a drying agent, at room temperature or in a refrigerator. Photographic development is done in Microdol, or in a special fine grain "physical" developer. For examination in the electron microscope the sections are stained with uranyl or lead stains. These methods give a good localization of the label, at the subcellular level, and good reproducibility in relative grain counts.     

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.     

Autometallography: tissue metals demonstrated by a silver enhancement kit

In biological tissue, minute accumulations of gold, silver, mercury and zinc can be visualized by a technique whereby metallic silver is precipitated on tiny accumulations of the two noble metals, or on selenites or sulphides of all four metals. In the present study a silver enhancement kit, primarily intended for the amplification of colloidal gold particles, has been used to demonstrate these catalytic tissue metals. Sections from animals exposed intravitally to aurothiomalatate, silver lactate, mercury chloride, sodium selenite or perfused with sodium sulphide were subjected to a commercial silver enhancement kit (IntenSE, Janssen Pharmaceutica). It was found that the kit performs adequately to the silver lactate gum arabic developer and to the photographic emulsion technique. The kit can be used as a silver enhancement medium for the demonstration of zinc by the Neo-Timm and selenium methods and for demonstration of gold, silver, and mercury in tissues from animals intravitally exposed to these metals. It can also be used for counterstaining silver treated osmium fixed tissues embedded in plastic.     

Histochemical demonstration of heavy metals

Summary The three steps of the sulphide silver method have been examined: 1) Transformation of metals to metal sulphides; 2) Fixation and embedding or freezing of the tissue for sectioning; and 3) Deposition of metallic silver on the metal sulphides in a physical developer. Based on the results, a revised method is described and discussed. It is particularly important 1) To maintain a sufficient but low concentration of sulphide ions during the perfusion; 2) To avoid using oxidating or acid fixatives; 3) To ensure low temperatures while embedding in paraffin or during polymerization of Epon; and 4) to use a slow-acting physical developer. Examples of the metal sulphide pattern from various tissues are presented.     

Ultrastructural autometallography: a method for silver amplification of catalytic metals

The autometallographic technique involves application of a silver bromide-containing emulsion on the surface of ultrathin sections placed on grids that are subsequently exposed to a photographic developer. In tissue sections from animals treated intravitally with gold, silver, or mercury compounds, accumulations of the metals are visualized by autometallography and can be used for quantitative studies. After amplification, sections can be stained with lead citrate and uranyl acetate. Using autometallography, particles of colloidal gold dispersed in a film of gelatin showed a time-dependent growth and were gradually amplified up to 3.5-fold after 15 min of development. Hence the method may prove useful tracing colloidal gold particles in sections with low particle density, and be a powerful tool for revealing metals in biological tissues.     

High resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary

The secretory process in pituitary mammotrophs was analyzed by quantitative electron microscope autoradiography. Dispersed pituitary cells from estrogen-treated female rats were subjected to pulse- labeling with [3H]leucine (5 min) followed by a chase incubation of up to 4 h. Autoradiograms were prepared using fine-grained emulsion (Kodak 129-01), and analyzed using a three-step "mask analysis' procedure: (a) the distribution of autoradiographic grains is determined as in a simple grain density analysis; (b) masks (transparent overlays) are used to generate expected grains from assumed sources; and (c) a computer program compares these two distributions and varies the expected distribution to match the observed distribution, thereby identifying the radioactive sources in the tissue. The overall route of intracellular transport of prolactin from rough endoplasmic reticulum (ER) leads to Golgi complex leads to immature secretory granules leads to mature secretory granules was as established in previous studies. However, by use of the high resolution emulsion and method of analysis, the precision with which label could be localized within individual source compartments was much greater and the time resolution was much sharper than achieved previously using Ilford L4 emulsion and simple grain density analysis. The main new findings were as follows: (a) the ER was essentially drained of radioactivity by 30 min, the Golgi complex by 1 h, and the immature secretory granules by 2h postpulse. This indicates that the secretory product (prolactin) is rapidly and efficiently transported out of these compartments. (b) approximately 30% of the total radioactivity remains located in the ground cytoplasm over the entire postpulse period examined (up to 4 h), and by 30 min postpulse the grain density in the ground cytoplasm exceeded that of the ER. This indicates the ability to resolve ER-associated label (presumably associated mainly with secretory products) from the cytoplasmic label (presumably associated with nonsecretory proteins). (c) the specific activity of immature secretory granules was much greater than previously appreciated; at 1 h postpulse it was greater than 200 times that of the adjacent Golgi complex cisternae. This large dynamic range in observed grain density demonstrates the ability to effectively correct for radiation spread and thus to detect with great accuracy high concentration of label even from very small structures (20-100 nm) which constitute a small percentage (less than 1%) of the total cell area.     

Proteomic identification of molecular targets of gambogic acid: Role of stathmin in hepatocellular carcinoma

Gamboge has been developed as an injectable drug for cancer treatment in China. In this study, the inhibition ratio and their IC₅₀ values of two derivatives from Gamboge in hepatocellular carcinoma (HCC) were determined. Proteomic approach was employed to reveal the target proteins of these two derivatives, gambogic acid (GA), and gambogenic acid (GEA). HCC cells were cultured under varied conditions with the addition of either GA or GEA. Twenty differentially expressed proteins were identified and the four most distinctly expressed proteins were further validated by Western blotting. GA and GEA revealed inhibitory effects on HCC cell proliferation. The expression of cyclin‐dependent kinase 4 inhibitor A and guanine nucleotide‐binding protein β subunit 1 were upregulated by both xanthones, whilst the expression of 14‐3‐3 protein sigma and stathmin 1 (STMN1) were downregulated. Furthermore, overexpression of STMN1 in HCC cells decreased their sensitivity, whilst small interfering RNAs targeting STMN1 enhanced their sensitivity to GA and GEA. In conclusion, our study suggested for the first time that STMN1 might be a major target for GA and GEA in combating HCC. Further investigation may lead to a new generation of anticancer drugs exerting synergistic effect with conventional therapy, thus to promote treatment efficacy.     

Binding analysis of glycyrrhetinic acid to human serum albumin: fluorescence spectroscopy, FTIR, and molecular modeling

Fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), and molecular modeling methods were employed to analyze the binding of glycyrrhetinic acid (GEA) to human serum albumin (HSA) under physiological conditions with GEA concentrations from 4.0x10(-6) to 4.5x10(-5) mol L(-1). The binding of GEA to HSA was via two types of sites: the numbers of binding site for the first type was near 0.45 and for the second type it was approximately 0.75. The binding constants of the second type binding site were lower than those of the first type binding site at corresponding temperatures, the results suggesting that the first type of binding site had high affinity and the second binding site involved other sites with lower binding affinity and selectivity. The fluorescence titration results indicated that GEA quenched the fluorescence intensity of HSA through static mechanism. The FTIR spectra evidence showed that the protein secondary structure changed with reduction of alpha-helices about 26.2% at the drug to protein molar ratio of 3. Thermodynamic analysis showed that hydrogen bonds were the mainly binding force in the first type of binding site, and hydrophobic interactions might play a main role in the second type of binding site. Furthermore, the study of computational modeling indicated that GEA could bind to the site I of HSA and hydrophobic interaction was the major acting force for the second type of binding site, which was in agreement with the thermodynamic analysis.     

Silver enhancement of lectin-gold and enzyme-gold cytochemical labelling of eggs of the nematode Onchocerca gibsoni

Summary Wheat germ agglutinin—gold and chitinase—gold complexes were used to demonstrate the presence of chitin on the surfaces of eggs of the animal parasitic nematodeOnchocerca gibsoni. The gold complexes were enhanced by silver intensification and examined by light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Distinctive labelling of the egg surfaces was obtained with both probes in all three microscope modes. The results indicate that the small colloidal gold markers (3–10 nm) commonly used for high resolution TEM studies may be silver enhanced and also used for sensitive LM and SEM studies.     

Processing Stripping Film Autoradiographs of Citrus Buds; Avoidance of Entrapped Air between Tissues and Developed Emulsion

Autoradiography was used on paraffin sections for investigating DNA synthesis in vegetative and flower buds of Citrus sinensis (L.) Osbeck. We preferred stripping film to liquid emulsion to obtain a more uniform thickness of the silver grain layer, as this would not vary more than ±10%. Furthermore, Baserga and Nemeroff (1962) have observed a lower sensitivity of fluid emulsion in comparison to stripping film together with occasionally erratic grain count and a slightly higher incidence of mechanical fogging. However, on using the customary procedure it was found that close contact between the surface of the section and the developed emulsion was not maintained when dehydration and clearing was done in the usual manner. The following modifications were therefore introduced to prevent the formation of air pockets between these two layers.     

Histochemical investigations on the nature of large blood vessel endothelial and medial argyrophilic lines and on the mechanism of silver staining

Summary A study of the mechanisms involved in silver staining of blood vessels has been performed on the rabbit and rat aorta and vena cava, both in fixed and unfixed states. Pretreatment with cationic detergents, organic solvents, and solutions containing free iodide ions inhibited the silver staining. Anionic or neutral detergents, oxidizing agents, binders of such ions as Ca⁺⁺, Mg⁺⁺ and SO ₄ ^- failed to inhibit the staining. Staining of the intercellular gaps between endothelial cells and between smooth muscle cells could also be obtained if vessels were treated with a cationic detergent and bromocresol green, or by a modified Hale's colloidal iron technique. Silver lines could be returned to dechlorinated vessels, if treated with sodium chloride before silver nitrate staining, but not vice versa; by an extended treatment with dilute silver nitrate or with gold chloride following normal silver nitrate staining; and by treatment with heparin prior to silver staining. Dark chamber experiments have demonstrated that a photographic developer can take the place of light in the silver staining procedure and that a photographic fixer has the same effect on vessel silver staining as dechlorination.The obtained results have led to the hypothesis that silver staining of vessels occurs in two stages. In the first silver ions from silver nitrate are bound by polyanions located primarily in the intercellular gaps, and then reduced. This produces a network of reduced silver grains which, however, are still too sparsely aggregated to be visualized. Chloride ions in the tissues also bind and precipitate silver ions preventing their removal in subsequent rinsing procedures. In the second stage light (or a photographic developer) reduces the silver ions in silver chloride, producing a visible accumulation of metallic silver, but only around the silver grains reduced during the first stage, analogous to the photographic process.The possible existence and function of an intercellular cement substance is discussed in light of the evidence for the presence of polyanionic groups in the intercellular gaps.     

Silver enhancement of quantum dots resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, in vitro and immersion created zinc-sulphur/zinc-selenium nanocrystals, (3) metal ions liberated from metal implants and particles

Autometallographic (AMG) silver enhancement is a potent histochemical tool for tracing a variety of metal containing nanocrystals, e.g. pure gold and silver nanoclusters and quantum dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium. These nanocrystals can be created in many different ways, e.g. (1) by manufacturing colloidal gold or silver particles, (2) by treating an organism in vivo with sulphide or selenide ions, (3) as the result of a metabolic decomposition of bismuth-, mercury- or silver-containing macromolecules in cell organelles, or (4) as the end product of histochemical processing of tissue sections. Such nano-sized AMG nanocrystals can then be silver-amplified several times of magnitude by being exposed to an AMG developer, i.e. a normal photographic developer enriched with silver ions. The present monograph attempts to provide a review of the autometallographic silver amplification techniques known today and their use in biology. After achieving a stronghold in histochemistry by Timm's introduction of the "silver-sulphide staining" in 1958, the AMG technique has evolved and expanded into several different areas of research, including immunocytochemistry, tracing of enzymes at LM and EM levels, blot staining, retrograde axonal tracing of zinc-enriched (ZEN) neurons, counterstaining of semithin sections, enhancement of histochemical reaction products, marking of phagocytotic cells, staining of myelin, tracing of gold ions released from gold implants, and visualization of capillaries. General technical comments, protocols for the current AMG methods and a summary of the most significant scientific results obtained by this wide variety of AMG histochemical approaches are included in the present article.     

Processing Stripping Film Autoradiographs of Citrus Buds; Avoidance of Entrapped Air between Tissues and Developed Emulsion

Autoradiography was used on paraffin sections for investigating DNA synthesis in vegetative and flower buds of Citrus sinensis (L.) Osbeck. We preferred stripping film to liquid emulsion to obtain a more uniform thickness of the silver grain layer, as this would not vary more than ±10%. Furthermore, Baserga and Nemeroff (1962) have observed a lower sensitivity of fluid emulsion in comparison to stripping film together with occasionally erratic grain count and a slightly higher incidence of mechanical fogging. However, on using the customary procedure it was found that close contact between the surface of the section and the developed emulsion was not maintained when dehydration and clearing was done in the usual manner. The following modifications were therefore introduced to prevent the formation of air pockets between these two layers.     

Chemical Stabilization of Golgi Silver Chromate Impregnations

Blocks of neural tissue were processed by a modified Golgi-Kopsch procedure and by the rapid Golgi method. Following the impregnation, the blocks were embedded in celloidin, sectioned at 100μm, and collected in 70% alcohol. The sections were then processed as follows: 1) rinsed in distilled water; 2) substituted with 0.4M sodium bromide for five minutes; 3) reduced in Kodak D-19 developer; and 4) treated in 0.5M sodium thiosulfate. The silver chromate deposits within the impregnated cells are converted successively to silver bromide and to reduced silver by this procedure. Sections so treated resist decomposition of the Golgi impregnation, and they may be counterstained with conventional aqueous cresyl violet to demonstrate the cytoarchitecture of the Golgi-impregnated tissue.