N A N O P R O B E S     E - N E W S

Vol. 8, No. 6          June 30, 2007

You can use FluoroNanogold for the following applications:

• Correlative fluorescence and electron microscopy. The absence of intervening staining steps ensures highest possible correlation.
• check labeling by fluorescence to make sure that it is successful before processing for electron microscopy.
• Confirm a staining pattern found in a previous immunofluorescence experiment for immunogold studies.

FluoroNanogold fluorescent and gold probes have been used for a number of different correlative microscopy methods:

• Combined and Correlative fluorescence and transmission electron microscopic (TEM) labeling.
• Correlative fluorescence and scanning electron microscopy (SEM).
• Correlative optical and X-ray fluorescence microscopy.

The illustration below shows the structure of FluoroNanogold and some results, including an example of combined fluorescent and SEM labeling.

Our Alexa Fluor®* FluoroNanogold probes offer the best possible fluorescence labeling performance in these applications:

• Increased fluorescence brightness and higher quantum yield.
• Improved solubility: lower background signal and higher signal-to-noise ratios.
• Fluorescence remains high and consistent across a wider pH range.

Since we now offer both Alexa Fluor®* 488 and Alexa Fluor®* 594 FluoroNanogold, you can now use these probes to differentiate multiple targets using different colored fluorescence.

Dynamic repositioning of telomeres, which is unique to meiotic prophase I and highly conserved among eukaryotes, has been shown to be required for proper alignment and recombination of homologous chromosomes. At the start of meiosis, telomeres attach to the nuclear envelope and transiently cluster in a limited area to form a chromosomal bouquet; however, the mechanism of this process is largely unknown. Schmitt and co-workers, as part of their investigation into this process, used FluoroNanogold to correlate immunoelectron microscopy with immunofluorescence in their recent study of the role of Sun2 in tethering mammalian meiotic telomeres to the nuclear envelope; their results are described in Proceedings of the National Academy of Sciences of the USA.

Cryosections or cells from testes suspensions from male Wistar rats were fixed for 5 minutes in phosphate-buffered saline (PBS) with 1% formaldehyde, permeabilized for 10 minutes in PBS containing 0.1% Triton X-100,and blocked for 1 hour with PBT (PBS containing 1.5% bovine serum albumin (BSA) and 0.1% Tween-20). Slides were then incubated simultaneously with selected primary antibodies for 20 minutes, washed twice in PBS, and bound antibodies were detected with corresponding secondary antibodies, either immunofluorescent, or FluoroNanogold. After FluoroNanogold labeling, the cryosections were washed in PBS, refixed for 10 minutes in 2% glutaraldehyde in PBS, washed several times in distilled water and silver enhanced.

The authors found that the SUN-domain protein Sun2 specifically localizes to the nuclear envelope attachment sites of meiotic telomeres: association is maintained throughout the dynamic movement of telomeres and does not require the assembly of chromosomal axial elements or the presence of A-type lamins. Electron microscopic analysis using antibodies against Sun2 and comparison with localization of its other components revealed that Sun2 is part of a membrane-spanning fibrillar complex that interconnects attached telomeres with cytoplasmic structures. These results, together with other studies on yeasts, show that the molecular mechanisms for tethering meiotic telomeres and their dynamic movements during bouquet formation are conserved among eukaryotes.

Reference:

• 1: Schmitt, J.; Benavente, R.; Hodzic, D.; Hoog, C.; Stewart, C. L., and Alsheimer, M.: Transmembrane protein Sun2 is involved in tethering mammalian meiotic telomeres to the nuclear envelope. Proc. Natl. Acad. Sci. USA, 104, 7426-7431 (2007).
  • Abstract (courtesy of Proceedings of the National Academy of Sciences of the USA).

One of the challenges in using combined fluorescent and gold probes is that the optimum use conditions (concentration and buffer) for the two labels may be slightly different, and some compromise may be required to find the conditions that give the best results. Control of non-specific binding may need to be quite rigorous, since both labels can interact non-specifically with biological components.

We have found that the following methods may help ensure the brightest, cleanest signal:

• The most effective blocking agent we have tested is 5% nonfat dried milk. This was particularly effective when it was used in the FluoroNanogold conjugate incubation buffer in addition to the blocking step. Cold-water fish gelatin has also been found to be helpful for gold probes generally.

• Adjusting camera exposure: manual control of exposure can help obtain the best signal-to-noise ratio. FluoroNanogold is frequently compared with commercially available fluorescently labeled IgG conjugates. Since these are larger and more highly labeled, they give brighter fluorescence. If automatic exposure adjustment is allowed with FluoroNanogold-stained specimens, the greater exposure can lead to higher apparent backgrounds. Setting the camera exposure manually can be used to overcome this effect.

• For reducing the background in electron microscopy, sodium citrate buffer was found to be more effective than other buffers when used as a wash before silver enhancement. 0.02 M sodium citrate at pH 7.0 works well with HQ Silver, while pH 3.5 works best with the Danscher silver formulation.

• Background binding is often attributed to hydrophobic interactions (both the gold and fluorescent labels have some hydrophobicity), and therefore adding reagents that reduce hydrophobic interactions to the wash buffer may help remove non-specific binding. Examples include:
  • 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling carbon dioxide into an aqueous suspension of triethylamine with stirring (Reference: Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77-91 (1986)).
  • 0.1% to 1% detergent, such as Tween-20, or Triton X-100. 0.1% saponin may also be useful since its effects are reversible, so ultrastructural preservation may be improved if it is removed in later steps.
  • 0.1% to 0.5% of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane.

More information:

• Catalog information: FluoroNanogold
• Alexa Fluor® 488 FluoroNanogold-Fab': Product information and instructions
• Alexa Fluor® 594 FluoroNanogold-Fab': Product information and instructions
• FluoroNanogold: Technical help
• FluoroNanogold: References, sorted by application

* Alexa Fluor is a registered trademark of Invitrogen - Molecular Probes

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Eliminating Background staining with Nanogold® and Silver Enhancement

Non-specific signal can arise from non-specific binding of an unlabeled primary antibody or probe, from a gold-labeled probe sticking to other parts of the system, or from the reaction of silver or gold enhancement reagents with other components of your specimen. A general approach which is often helpful is to remove each component in turn from your system, and compare the effect on background signal:

• Omit the primary antibody and apply the gold- or FluoroNanogold-labeled secondary and (if you use it) silver enhance as usual. If the background disappears, it may be due to the primary antibody. In this case, reducing the concentration of primary antibody or using more thorough washing or permeabilization after incubation may help reduce background. Another potential source of background is the presence of traces of amine-reactive fixatives, such as glutaraldehyde, which can react with proteins: these should be quenched with glycine, ammonium chloride, or sodium cyanoborohydride before immunostaining.

• Omit the gold-labeled secondary, but apply the primary and silver or gold enhancement reagents as usual. If this fixes the problem, then it may be due to non-specific binding by the gold conjugate. In this case, solutions should address the mechanisms by which gold might adsorb to components of the specimen:
  • 5 % nonfat dried milk has been found to be highly effective in reducing FluoroNanogold and Nanogold background. This was found to be particularly effective when mixed with and added to the specimen together with the FluoroNanogold conjugate.

  • Background binding is often attributed to hydrophobic interactions (both gold and fluorescent labels have some hydrophobicity). Adding reagents that reduce hydrophobic interactions to the wash buffer or the gold conjugate incubation buffer may reduce non-specific binding. Examples include 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling carbon dioxide into an aqueous suspension of triethylamine with stirring. Reference: Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77 (1986)); 0.1 % to 1 % detergent, such as Tween-20, or Triton X-100; and 0.1 % to 0.5 % of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane.

  • Does the distribution of the binding suggest a blocking method? For example, if it occurs in thiol (cysteine)-rich regions, it may be due to thiol coordination to the gold. This may be blocked using N-ethylmaleimide. If it occurs in nuclear material, it may be due to interactions with the ionic charges of nucleic acids; increasing the ionic strength of the buffer, or changing the pH to a value at which the nucleic acids are less ionized, may help.

  • With FluoroNanogold, manual camera exposure can help in reducing fluorescence background. FluoroNanogold is frequently compared with commercially available fluorescently labeled IgG conjugates, which are larger and more highly labeled and give brighter fluorescence. Automatic exposure adjustment with FluoroNanogold-stained specimens can result in greater exposure and higher apparent backgrounds: Setting the camera exposure manually can be used to overcome this effect.

  • A full range of solutions has been discussed in previous articles for removing background due to Nanogold and FluoroNanogold.

• If neither approach reduces background, it maybe due to the autometallographic reagent - the silver or gold enhancement step. However, it may also be due to changes in tissue processing, blocking, washing or fixation; it may be worthwhile checking to see whether any changes have been made in these that could lead to background. Effective fixes for background caused by silver or gold enhancement are those that address the redox chemistry of these reagents:
  • Wash samples with a chelating agent before silver enhancement to sequester any redox-active transition metals that can catalyze silver enhancement. Suitable reagents include 0.02 M sodium citrate buffer: adjust to pH 7.0 if you are working with Danscher silver enhancer or your development is too slow, and adjust to pH 3.5 if you are working with HQ Silver or your development is too fast. (Reference: Powell, R. D.; Halsey, C. M.; Spector, D. L.; Kaurin, S. L.; McCann, J., and Hainfeld, J. F.: A covalent fluorescent-gold immunoprobe: simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy. J. Histochem. Cytochem., 45 947-956(1997)). Another useful reagent is disodium EDTA (0.05 M, pH 4.6) - use as the last wash before adding the silver enhancement reagent. These reagents also help ensure a uniform sample pH so that development times are consistent.

  • Ensure that all samples are thoroughly washed with ultrapure or deionized water before silver enhancement to remove halide ions: these will react with any silver salts to form a precipitate, which is visible in the EM and will nucleate additional background staining.

  • Use plastic or teflonized (not metal) forceps and tools for handling specimens.

• If all else fails, use a stop or back-development step after silver or gold enhancement to prevent the reaction from continuing within the specimen after rinsing, or if necessary to remove excess silver or gold. Methods include:
  • Apply freshly prepared 1% sodium thiosulfate solution for one minute (or longer) until the excessive silver or gold deposits are removed.

  • Treat with Lugol's iodine for 30 seconds to one minute, then remove the residual orange-brown color of the iodine using freshly prepared 1% sodium thiosulfate solution.

  • Back develop with Farmer's Solution (0.3 ml 7.5% potassium ferricyanide, 1.2 ml of 20% sodium thiosulfate, 60 ml water).

• A full range of solutions has been discussed in previous articles for removing background due to silver enhancement and gold enhancement.

Lot to lot variations in any of these components can also cause background, as well as differences in specimen preparation, washing, and blocking or fixation reagents. The best strategy for finding the source in this case is to replace each component in turn with an equivalent from a different lot; the reagent causing the background is the one whose replacement solves the problem.

More information:

• Tech help: reducing background with Nanogold® labeling
• Tech help: controlling background with silver enhancement
• Controlling background - FluoroNanogold
• Keeping GoldEnhance under control
• Getting the best out of NTA-Ni(II)-Nanogold

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HQ Silver Gives Better Results with All Types of Gold

• It is the only commercial silver enhancement reagent that includes a thickening protective colloid to ensure uniform, controlled development. This produces uniform particle size.

• Once mixed, the solution has a pH relatively close to neutral, and a very low ionic strength. These ensure a high level of ultrastructural preservation, making the reagent compatible with all levels of fixation.

• HQ Silver is formulated to ensure that the highest proportion of gold particles are enlarged, making it ideal for quantitative immunolabeling studies.

HQ Silver works equally well on all types of gold particles, including those from other suppliers. It has provided superior results with colloidal gold, with our own Nanogold conjugates, and with other types of gold particles, giving consistent, uniform enlargement of a high proportion of gold particles.

HQ Silver produces excellent results in pre-embedding immunogold labeling, especially with Nanogold®. However, in their paper in Neuroscience Letters, Dumartin and group confirmed that it is also highly effective for enhancing ultrasmall gold; their work follows a recent study of dopamine receptor distribution and function which used a similar method.

D1 type receptors (D1R) are highly expressed in the dorsal striatum and nucleus accumbens. In the dorsal striatum, they are rarely observed on presynaptic terminals; the authors wished to compare their subcellular localization in the nucleus accumbens core and shell with that in dorsal striatum. To do so, they used a pre-embedding immunogold method using ultrasmall (0.8 nm) gold with silver enhancement to localize D1 receptors in Sprague-Dawley rat brain sections.

After anesthesia with chloral hydrate and transcardial perfusion with 50100 ml of 0.9% sodium chloride and 300 ml of fixative (2% paraformaldehyde and 0.2% glutaraldehyde in 0.1M phosphate buffer, pH 7.4), the brains were removed, stored in 2% paraformaldehyde overnight, then cut into 60 µm frontal sections. The sections were collected, cryoprotected (PBS containing 25% saccharose and 10% glycerol) and freeze-thawed in isopentane to improve immunoreagent penetration.

D1R immunoreactivity was detected at the electron microscopic level by pre-embedding immunogold labeling using a monoclonal antibody raised in rat against a 97 amino acid sequence corresponding to the C terminus of the human D1R: vibratome sections were incubated in 4% normal goat serum (NGS) for one hour, then in D1R antibody (1:1000) supplemented with 1% NGS overnight at room temperature (RT). After washing twice in PBS and twice in PBS-acylated bovine serum albumin with 0.1% fish gelatin (PBS-BSAc-gel), the sections were incubated for 4 hours at RT in goat anti-rat IgG - ultrasmall gold conjugate diluted (1:100) in PBS-BSAc-gel. After washing (3 x PBS), postfixation (1% glutaraldehyde in PBS for 10 minutes) and washing again (2 x PBS; 2 x sodium acetate buffer, 0.1M, pH 7.0), the immunogold signal was intensified with HQ silver for 710 minutes at RT in the dark. The reaction was stopped by two washes in sodium acetate.

After several washes in PBS, the sections were postfixed in 0.50% osmium tetroxide for 10 minutes, dehydrated in ascending series of ethanol dilutions that included 70% ethanol containing 1% uranyl acetate, treated with propylene oxide, and impregnated in resin overnight. Sections were mounted on glass slides, flat-embedded in resin and cured for 48 hours at 60°C. Areas of interest were cut out from the same vibratome sections and glued to blank cylinders of resin; control semi-thin immunostained sections (1 µm-thick) were collected on glass slides, dried and mounted in Eukitt. Serial ultrathin immunostained sections were cut, collected on pioloform-coated single slot grids, contrasted with lead citrate and observed by transmission electron microscopy.

Among all presynaptic terminals forming asymmetric contact with dendritic processes, the percentage of D1R immunoreactive terminals was low in the dorsal striatum (8.2%), but significantly higher in the nucleus accumbens core and shell at 25.5% and 29% respectively. This is consistent with electrophysiological studies, in which D1 stimulation inhibited the response of target neurons to glutamatergic input via presynaptic mechanisms in the nucleus accumbens, but not in the dorsal striatum.

Reference:

• Dumartin, B.; Doudnikoff, E.; Gonon, F., and Bloch, B.: Differences in ultrastructural localization of dopaminergic D1 receptors between dorsal striatum and nucleus accumbens in the rat. Neurosci. Lett., 419, 273-277 (2007).
  • Abstract (courtesy of Science direct).

More information:

• Silver Enhancers: Catalog information
• HQ Silver: Product information, instructions and protocols
• Technical help: Silver enhancement
• Silver enhancement: References, sorted by application
• Application feature: Nanogold pre-embedding immunolabeling with HQ Silver enhancement

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Undecagold: The Smallest Commercial Gold Label

• STEM microscopy.
• Image analysis.
• Diffraction studies.
• Heavy atom derivatization of large proteins and membrane proteins in crystallography.
• As a high-resolution microscopy size standard.

Although undecagold is not as responsive to silver enhancement as the larger Nanogold®, it may still, as Flierl and co-workers reported be successfully silver-enhanced for electron microscope observation; They used undecagold labeled PNA-Peptide/Oligonucleotide (PPO)-Complexes to demonstrate a novel method for importing DNA into mitochondria in living cells, an application for which the small size proved critical. Now, Girard and co-workers, in a recent paper in Life Sciences report the use, with silver enhancement, of a 0.8 nm gold-labeled streptavidin acquired from Nanoprobes, which they used to investigate the localization and activity of butyryl cholinesterase (BChE) in diaphragm and the tibialis anterior muscle from perfused mice.

At the neuromuscular junction (NMJ), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can both hydrolyze acetylcholine (ACh); the released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft, where pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. However, little is known about butyryl cholinesterase (BChE). AChE knockout mice (AChE-KO) provide a valuable tool for examining the role of BChE in the absence of AChE activity. AChE-KO mice show an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function.

Pre-embedding immunogold labeling with silver enhancement was used to investigate the distribution of BChE within neuromuscular junctions. The diaphragm and tibialis of perfused mice was processed for electron microscopy. Junctions were first identified by detecting nicotinic AChRs using biotinylated alpha-bungarotoxin, using a pre-embedding immunogold method. Muscle fibers were incubated in biotinylated alpha-bungarotoxin (10 µg/mL in phosphate buffered saline (PBS)) for 3 days at 4°C. After washing, muscles were incubated for 2 hours in streptavidin coupled to undecagold. The fibers were then washed, postfixed in 1% glutaraldehyde for 10 minutes, washed again in acetate buffer (0.1 M, pH 7), and silver enhanced with HQ silver for 2 minutes at room temperature in the dark. After a further wash in acetate buffer, the sections were treated with 1% osmium, dehydrated and embedded in resin. Ultra thin sections were cut, stained with lead citrate and examined in the transmission electron microscope.

Analysis of the electron microscopic results showed that is present at the endplate region of wildtype and AChE-KO mature muscles. Electrophysiological measurements indicated that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release may explain the very high sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that the role of BChE at the NMJ is to protect nerve terminals from an excess of ACh.

While we are always pleased to see publications citing our products, we have to wonder about this one. We have not sold undecagold as a streptavidin conjugate for several years, and the general experimental procedure sounds more like Nanogold than undecagold. If you think undecagold may be right for you, we suggest that you check the Flierl paper as well for a good overview.

References:

• Girard E.; Bernard V.; Minic J.; Chatonnet A.; Krejci E., and Molgo, J.: Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice. Life Sci., 80, 2380-2385 (2007).
  • Abstract (courtesy of Science Direct).

• Flierl, A.; Jackson, C.; Cottrell, B.; Murdock, D.; Seibel, P., and Wallace, D. C.: Targeted delivery of DNA to the mitochondrial compartment via import sequence-conjugated peptide nucleic acid. Mol. Ther., 7, 550-557 (2003).
  • Abstract (courtesy of PubMed (Medline)).

More information:

• Catalog information: Undecagold reagents
• Instructions and product information: Monomaleimido Undecagold
• Instructions and product information: Mono-Sulfo-NHS Undecagold
• Technical help: undecagold reagents
• Undecagold references, sorted by application

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Nanoprobes at Microscopy & Microanalysis 2007

Our paper, number 848, entitled "Correlative Enzymatic and Gold Probes for Light and Electron Microscopy," will be presented in session B01B, "Structural analysis of biological systems: an integrative understanding of organellar, cellular, and organismal function," scheduled for Thursday, August 9, beginning 3:15 PM in room 222. Microscopy & Microanalysis 2007 will be held in Broward County Convention Center in Fort Lauderdale, Florida.

Nanoprobes will be closed on Wednesday, July 4 in observance of the United States Independence Day holiday.

More information:

• Microscopy & Microanalysis 2007
• Microscopy & Microanalysis 2007 - Abstract search engine
• Scientific Program (from Microscopy & Microanalysis, Cambridge University Press)
• Previous Microscopy & Microanalysis presentations on combined fluorescent and gold probes
• Nanoprobes news and press releases

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Other Recent Publications

Reference:

• Chen, J.; Wang, D.; Xi, J.; Au, L, Siekkinen, A.; Warsen, A.; Li, Z. Y.; Zhang, H.; Xia, Y., and Li X.: Immuno gold nanocages with tailored optical properties for targeted photothermal destruction of cancer cells. Nano Lett., 2007 7, 1318-1322 (2007).
  • Abstract (courtesy of ACS Publications).

A long-running problem with immunohistochemistry is the difficulty in differentiating multiple targets when the antibodies are raised in the same species; this is frequently an obstacle because many of the antibodies used are raised in rabbits. However, Tóth and Mezey, in their recent paper in the Journal of Histochemistry and Cytochemistry, present a solution using tyramide signal amplification with different fluorescent tyramides for each staining step, and an intervening microwave treatment between each staining. Previously reported protocols addressing this issue all have serious limitations in terms of the time required, the sample format, or tissue degradation between each staining procedure. The authors eliminate cross-reactivity by using a tyramide signal amplification reaction, in which a horseradish peroxidase conjugate is used to catalytically deposit a labeled tyramide at the target site. The intervening microwave treatment is formulated to eliminate both immunoreactivity and peroxidase reactivity: therefore, a second target can be visualized with a rabbit antibody without interference either from cross-reaction with the primary antibody or peroxidase reactivity from the preceding step. This provides a simple, quick, and inexpensive solution which has two major advantages over existing methods. First, two or more antigens can be visualized in the same section with commercially available fluorescent dyes. Second, because of the high sensitivity of the tyramide signal amplification reaction, the method can be used to visualize both rare and abundant antigens, or multiple rare antigens.

Reference:

• Tóth, Z. E., and Mezey, E.: Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. J. Histochem. Cytochem., 55, 545-554 (2007).
  • Abstract (courtesy of Journal of Histochemistry and Cytochemistry).

On the subject of multiple staining, Kobayashi and group report the simultaneous labeling of five different lymphatic basins using quantum dots of different colors, in their Nano Letters paper. Quantum dots can be used to perform multicolor images with high fluorescent intensity, and are the right size for lymphatic imaging via direct interstitial injection. The authors used five carboxyl-functionalized quantum dots. All were established to be between 16 and 19 nm in mean size by dynamic light scattering, TEM and HPLC, but showed different emission spectra: three cadmium-selenium (Cd-Se; 565, 605, and 655 peak emission) and two cadmium-tellurium (Cd-Te; 705 and 800 peak emission). Ten-week-old normal athymic female mice were anaesthetized by intraperitoneal injection of 1.15 mg sodium pentobarbital; the five different quantum dots were then injected into five sites connecting to different lymph nodes, and imaged using wavelength-resolved spectral imaging. This allowed noninvasive, simultaneous visualization of five separate lymphatic flows draining; this approach and may be applicable to predicting the route of cancer metastasis into the lymph nodes.

Reference:

• Kobayashi, H.; Hama, Y.; Koyama, Y.; Barrett, T.; Regino, CA.; Urano, Y., and Choyke, P. L.: Simultaneous Multicolor Imaging of Five Different Lymphatic Basins Using Quantum Dots. Nano Lett., 7, 1711-1716 (2007).
  • Abstract (courtesy of ACS Publications).

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