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Updated: November 8, 2005

N A N O P R O B E S     E - N E W S

Vol. 6, No. 11          November 8, 2005


In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

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Validation of a Chromogenic Dual ISH and IHC Assay for HER2 Gene and Protein Status

Enzyme Metallography (EnzMet) has proven to be highly effective as a detection method for in situ hybridization. It detects single gene copies with extreme sensitivity and resolution, enabling clear counting of gene signals, and the black, punctate staining is readily visualized and differentiated from other stains in the conventional brightfield light microscope without the need for oil immersion.

In collaboration with Dr. Raymond Tubbs and group at the Cleveland Clinic Foundation, we now report that this method may be combined with a second staining protocol in order to visualize both HER2 gene copies and HER2 protein overexpression in the same slide. The HER2 protein is visualized using Fast Red K immunohistochemistry, providing a clear and visually distinct signal, and this method provides the pathologist with a way to compare gene and protein changes directly.

Breast cancer is a devastating illness, with an estimated 210,000 new cases diagnosed and 40,600 fatalities in 2003 in the US. Cancer is characterized by both genetic and proteomic aberrations, and the determination of both are important for diagnosis and therapy. An important factor in breast cancer malignancy, and in therapy selection, is the HER2 gene, which codes for a transmembrane tyrosine kinase growth factor receptor: amplification of this gene, and overexpression of the HER2 protein, are correlated with a significantly worse prognosis, greater likelihood of malignant behavior, and less favorable response to conventional therapy. HER2 gene or protein status is used to identify patients for whom Trastuzumab (Herceptin) therapy and its combinations would be beneficial. Both assessments play valuable roles in diagnosis, and the ongoing development of new combination therapies, combined with our increasingly complex understanding of the relationship between the prognostic factors and the variation in behavior between individual cancers, will create increasing demand for both of these, and other criteria, to be assessed at diagnosis.

A particular concern is distinguishing genuine amplification of a gene from polysomy, in which chromosome duplication results in more copies of the gene than usual. Differentiating the two requires counting the individual copies, and a clinically useful detection method must be both sufficiently sensitive to detect each gene copy, and sufficiently precise that it may be distinguished from its neighbors. The enzyme metallography method has ideal properties for this application.

[EnzMet GenePro Schematic (87k)]

left: The enzyme metallography process. right: schematic of the combined immunohistochemistry and in situ hybridization protocols used for the EnzMet GenePro assay, showing the localization of the targets and the different detection probes.

The study was performed on tissue, "midiarrays," consisting of 94 cores from 80 cases of invasive breast carcinoma, comprising 4 infiltrating lobular carcinoma, 1 mixed infiltrating ductal and lobular carcinoma, and 89 infiltrating ductal carcinoma; tissues were obtained from resection specimens or excisional biopsies from the Department of Anatomic Pathology at the Cleveland Clinic Foundation, after obtaining Institutional Review Board approval. All were neutral buffered formalin- fixed and paraffin-embedded. Whole tissue sections stained with hematoxylin and eosin were evaluated for infiltrating carcinoma and appropriate areas scored for tissue midiarray construction. Midiarrays are a large-core modification of conventional tissue microarrays, and consist of ten 3-mm-diameter tissue cores per slide, with a core of liver serving as a spatial anchor. After array construction, 4-mm sections were placed on electrostatically charged slides; the last section was stained with hematoxylin and eosin to confirm the presence of infiltrating carcinoma. Bright-field detection of HER2 gene amplification was achieved using enzyme metallography, and was combined with immunohistochemical (IHC) HER2 protein detection using alkaline phosphatase and fast red K substrate visualization. All components of the assay, including online deparaffinization and cell conditioning, were performed on an automated platform (Benchmark; Ventana Medical Systems) except for the hybridization step, which was performed off-line manually. For comparison, dual-color direct fluorescence in situ hybridization (FISH) (HER2/CEP17 PathVysion, Vysis, Downers Grove, IL) and IHC using monoclonal antibody CB11 (Ventana, dilution 1:40) were performed as described in previous publications.

The EnzMet ISH component of the assay was scored as either HER2 gene amplified (6 or more copies per nucleus), polysomic (3-5 copies per nucleus), or nonamplified (2 copies per nucleus); the IHC component was scored using the conventional FDA scale of 0 to 3+. Concordance of the EnzMet ISH component versus FISH was assessed and showed an excellent correlation (Pearson coefficient of 0.95; P , 0.001). The combination of gene and protein detection (EnzMet GenePro) displayed a specificity of 100% and an accuracy of 92.6% (95% confidence interval 85.397.0), facilitated recognition of gene/protein discordances, and allowed for efficient interpretation of the slide by conventional light microscopy. The interobserver kappa for each component was excellent (IHC, k = 0.94; and EnzMet, k = 0.96). EnzMet is the first bright-field ISH assay in our experience that routinely and nonambiguously detects endogenous HER2 signals, essential for a reliable clinical HER2 assay, and in combination with HER2 protein enables improved diagnosis in borderline cases.

References:

  • Downs-Kelly, E.; Pettay, J.; Hicks, D.; Skacel, M.; Yoder, B.; Rybicki, L.; Myles, J.; Sreenan, J.; Roche, P.; Powell, R.; Hainfeld, J.; Grogan, T., and Tubbs, R.: Analytical Validation and Interobserver Reproducibility of EnzMet GenePro: A Second-Generation Bright-Field Metallography Assay for Concomitant Detection of HER2 Gene Status and Protein Expression in Invasive Carcinoma of the Breast. Am. J. Surg. Pathol., 29, 1505-1511 (2005).

  • Tubbs, R.; Pettay, J.; Hicks, D.; Skacel, M.; Powell, R.; Grogan, T., and Hainfeld, J.: Novel bright field molecular morphology methods for detection of HER2 gene amplification. J. Mol. Histol., 35, 589594 (2004).

    More information:

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    Labeling other Functional Groups: Phosphates in Lipids

    Although we offer a selection of Nanogold$#174; and undecagold-labeled lipids, we are often asked about the feasibility of labeling other molecules of this type. Lipids are often particularly difficult candidates for Nanogold labeling because they usually do not contain many functional groups or other reactive points to which a Nanogold particle may be cross-linked. Take, for example, PI(3)P:

    [Molecular structure of PI(3)P (12k)]

    Structure of PI(3)P, showing the functional groups available for possible modification or cross-linking.

    Options for conjugating Nanogold to this molecule are very limited, because the PI(3)P molecule does not have any reactive functional groups to which Nanogold may be readily conjugated. Thiols or primary aliphatic amines would be best; however, as we have explained in previous articles, cross-linking chemistry is also possible for carboxylic acids or hydroxyls, although these procedures are slightly more difficult. However, PI(3)P has none of these, and contains only three groups which can be readily modified in any way. Is it possible to label any of them with Nanogold?

    Considering each group in turn:

    • The phosphate group: This is the best choice for labeling, because cross-linking chemistry has already been described in the literature. The simplest and best-documented approach is to activate the phosphate group in the same manner as a carboxylic acid: either by EDC/Sulfo-NHS coupling, activating with EDC (1-ethyl-3,3'-dimethylaminopropyl carbodiimide) followed by sulfo-NHS, or by treatment with CDI (N,N'-carbonyldiimidazole) in DMF (dimethylformamide). In either case, you could then react the activated ester with Monoamino-Nanogold.

      Reference:

      Chu, B. C. F.; Kramer, F. R., and Orgel, L. E.: Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res., 14, 5591-5603 (1986).

      Note that there is another option besides direct reaction of the activated ester with Monoamino Nanogold. The authors react their activated ester with cystamine, which is cleavable to give a thiol group: an alternative approach for Nanogold labeling is use cystamine exactly as described, cleave, and label using Monomaleimido Nanogold. Both reactions are described in more detail in the technical help section of our web site.

    • The double bonds: While modification of the double bonds is possible chemically, it is likely to be undesirable because modification of these groups may change the biological or liposomal behavior of the molecule.

    • The carbonyl (C=O) group: However, because this is actually part of an ester linkage, reactivity will be difficult and most methods will involve cleavage of the ester group, so part of the PI(3)P will be lost. Two potentially useful reactions are the Wittig or Horner-Wadsworth-Emmons reactions; however, these generally do not work well with esters, and you will probably need to find a variation that is adapted to this functionality. If your experiment would work with a cleaved derivative, this may be an option.

    The reactions used for phosphate group labeling are shown below:

    [Reaction scheme for Nanogold labeling of PI(3)P via the phosphate group (55k)]

    Reaction scheme illustrating labeling via a phosphate group, showing labeling of PI(3)P with Monoamino Nanogold or Monomaleimido Nanogold.

    One consideration in selecting functional group for labeling is whether the site participates in, or might hinder, the activity that you will be using the labeled probe to study; if so, then this may not be the best strategy. It is often preferable to conjugate Nanogold to the hydrophilic region of the molecule rather than the hydrophobic domain, both because Nanogold itself is hydrophilic, and also because the assembly of lipids into liposomes and other structures is mediated by the interactions of the hydrophobic domain: this activity would be impacted by the attachment of a large label at this site.

    More information:

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    Double and Triple Labeling with Silver-Enhanced Nanogold® and HRP

    Double labeling with silver-enhanced Nanogold® and HRP-DAB has generated a steadily growing body of publications, and Feng and co-workers have now extended the approach to triple labeling for their investigation into whether gamma aminobutyric acid (GABA) / Glycocontaining terminals might make synaptic contacts with neurokinin-1 receptor (NK1R)-expressing neurons in the sacral dorsal commissural nucleus (SDCN). Previous studies have shown that neurons in the SDCN express NK1R and can be modulated by the co-release of GABA and glycine (Gly) from a single presynaptic terminal; this suggests the possibility that GABA/Glycocontaining terminals might make synaptic contacts with NK1R-expressing neurons.

    In order to test this hypothesis, triple-immunohistochemical studies were performed in the SDCN. Triple-immunofluorescence histochemical studies showed that some axon terminals in close association with NK1R-immunopositive (NK1R-ip) neurons in the SDCN were immunopositive for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). Therefore, electron microscopic dual- and triple-immunohistochemistry was conducted for GAD/GlyT2, GAD/NK1R, GlyT2/NK1R, and GAD/GlyT2/NK1R.

    Spinal cords from rats perfused transcardially with 100 ml of 0.01M phosphate-buffered saline (PBS, pH 7.4), followed by 500 ml of 0.1M phosphate buffer (PB, pH 7.4) containing 4% (w/v) paraformaldehyde, 0.05% (w/v) glutaraldehyde and 15% (v/v) saturated picric acid were removed and placed in 0.1MPB (pH 7.4) containing 30% (w/v) sucrose overnight at 4°C. 50-micron frontal sections were collected consecutively in five series. After cryoprotection, sections were freeze-thawed in liquid nitrogen, placed in 0.05M Tris-buffered saline (TBS, pH 7.4) containing 20% normal goat serum (NGS) for 1 hour, then processed for electron-microscopic dual- and triple-immunohistochemistry. The first series of sections was used for double immunohistochemistry of GAD and GlyT2: these were incubated at room temperature sequentially with: (1) a mixture of 1 microgram/mL anti-GAD mouse IgG and 1 microgram/mL anti-GlyT2 guinea pig IgG for 24 hours; (2) a mixture of 10 microgram/mL biotinylated anti-[mouse IgG] donkey antibody (1:200) and Nanogold-IgG Goat anti-Guinea pig IgG (1:100).

    The second and the third series were processed for dual immunohistochemistry for GAD/NK1R or GlyT2/NK1R, respectively. The immunoperoxidase method for either GAD or GlyT2 was combined with the immunogold-silver method for NK1R. The sections were incubated at room temperature sequentially for GAD/NK1R with: (1) a mixture of 1 microgram/mL anti-GAD mouse IgG and 1 microgram/mL anti-NK1R rabbit IgG for 24 hours; and (2) a mixture of biotinylated anti-[mouse IgG] donkey antibody and Nanogold-IgG goat anti-rabbit IgG; for GlyT2/NK1R with: (1) a mixture of 1 microgram/mL anti-GlyT2 guinea pig IgG (Chemicon) and 1 microgram/mL anti-NK1R rabbit IgG for 24 hours, and (2) with a mixture of biotinylated anti-[guinea pig IgG] goat antibody (Vector) and Nanogold-IgG goat anti-rabbit IgG for 1618 hours. The fourth series of sections was processed for triple-immunohistochemistry for GAD/GlyT2/NK1R: the immunoperoxidase method for GAD was combined with the immunogold-silver method for GlyT2 and NK1R. The sections were incubated at room temperature sequentially with: (1) a mixture of 1 microgram/mL anti-GAD mouse IgG, 1 microgram/mL anti- GlyT2 guinea pig IgG and 1 microgram/mL anti-NK1R rabbit IgG for 24 h; and (2) a mixture of biotinylated anti-[mouse IgG] donkey antibody, Nanogold-IgG Goat anti-Guinea pig IgG and Nanogold-IgG anti-rabbit IgG for 1618 hours. Between steps (1) and (2), the sections were washed with 0.05M TBS. The incubation medium for all antibodies was 0.05M TBS containing 2% (v/v) normal goat serum (NGS). Subsequently, the sections were processed for EM by (1) silver enhancement with HQ Silver; (2) incubation with ABC Elite Kit (1:50; Vector) and visualization of GAD-IR or GlyT2-IR using diaminobenzidine (DAB); (3) osmification with 1% OsO4 in 0.1M PB (pH 7.4) for 1 hour; (4) counterstaining with uranyl acetate; and (5) flat embedding in Durcupan. Ultrathin sections through the SDCN were prepared, mounted on single-slot grids, and examined electron microscopically.

    In the EM triple immunohistochemistry for GAD/GlyT2/NK1, both GlyT2-IR and NK1R-IR were marked by silver grains. However, in the EM dual immunochemistry for GlyT2/NK1, GlyT2-IR was detected only within axon terminals, while NK1-IR was observed specifically within profiles of neuronal cell bodies and dendrites; therefore, although represented by the same signal, the two targets could be distinguished in the EM triple GAD/GlyT2/NK1-immunohistochemistry: silver grains within the axon terminals and those within the profiles of neuronal cell bodies and dendrite were considered to represent GlyT2-IR or NK1-IR, respectively.

    Axon terminals labeled with both electron-dense peroxidase immunoreaction products (GAD-ip) and silver grains (GlyT2-ip) were observed to be in symmetric synaptic contact with neuronal cell bodies or dendrites labeled with silver grains (NK1R-ip) within the SDCN. In total 45 synaptic axon terminals showing both GAD-IR and GlyT2-IR were observed upon NK1R-ip neuronal profiles within the SDCN; 39 (86.7%) of them were on dendritic profiles (Fig. 3D), and the remaining 6 (13.3%) on somatic profiles. On the basis of these results, it appears to be highly possible that some synaptic terminals upon NK1R-expressing SDCN neurons co-release GABA and Gly. The origins of the axon terminals co-expressing GAD- and GlyT2- immunoreactivity in the SDCN, however, still remain to be settled. The present results are in good accordance with the physiological data indicating that SDCN neurons might be modulated by presynaptic co-release of GABA and Gly.

    Reference:

    Feng, Y.-P.; Li, Y.-Q.; Wang, W.; Wu, S.-X.; Chen, T.; Shigemoto, R., and Mizuno, N.: Morphological evidence for GABA/glycine-cocontaining terminals in synaptic contact with neurokinin-1 receptor-expressing neurons in the sacral dorsal commissural nucleus of the rat. Neurosci. Lett., 18, 144-148.

    References describing methods in more detail:

    • Li, J. L.; Wang, D.; Kaneko, T.; Shigemoto, R.; Nomura, S., and Mizuno, N.: Relationship between neurokinin-1 receptor and substance P in the striatum: light and electron microscopic immunohistochemical study in the rat. J. Comp. Neurol., 418, 156163 (2000).

      Li, J. L.; Wu, S. X.; Tomioka, R.; Okamoto, K.; Nakamura, K.; Kaneko, T., and Mizuno, N.: Efferent and afferent connections of GABAergic neurons in the supratrigeminal and the intertrigeminal regions. An immunohistochemical tract-tracing study in the GAD67-GFP knockin mouse. Neurosci. Res., 51, 8191 (2005).

    More information:

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    Keeping GoldEnhance Under Control

    In addition to Kenzaka (reported last month), other users are discovering the benefits of our gold enhancement technology. Gold enhancement is an autometallographic method, similar to silver enhancement, in which gold is deposited onto gold nanoparticles. It has significant advantages for both scanning electron microscopy (SEM) and transmission electron microscopy (TEM):

    • Gold enhancement may safely be used before osmium tetroxide - it is not etched.
    • May be used in physiological buffers (chlorides precipitate silver, but not gold).
    • The metallographic reaction is less pH sensitive than that of silver.
    • Gold gives a much stronger backscatter signal than silver.
    • GoldEnhance is near neutral pH for best ultrastructural preservation.
    • Low viscosity, so the components may be dispensed and mixed easily and accurately.

    Alexander and co-workers show that gold enhancement works equally well with conventional colloidal gold in their recent paper in Proceedings of the National Academy of Sciences of the USA. They used a combination of immunofluorescence microscopy and SEM with gold-enhanced colloidal gold labeling to study the distribution and mobility of the sodium-hydrogen exchanger isoform 3 (NHE3), located on the apical membrane, which is fundamental to the maintenance of systemic volume and pH homeostasis. Proximal tubular reabsorption of filtered sodium is finely regulated by a variety of hormones and by changes in ionic composition and volume. The authors analyzed the subcellular distribution and dynamics of the exchangers in an epithelial line expressing NHE3 tagged with an exofacial epitope, which enabled monitoring exchanger mobility and traffic in intact cells.

    Using determinations of fluorescence recovery after photobleaching in combination with dynamic measurements of subcellular distribution, NHE3 was found to exist in four distinct subcompartments in renal epithelial cells: a virtually immobile subpopulation retained on the apical membrane by interaction with the actin cytoskeleton, in a manner that depends on the sustained activity of Rho GTPases; a mobile subpopulation on the apical membrane, which can be readily internalized; and two intracellular compartments that can be differentiated by their rate of exchange with the apical pool of NHE3. The authors sought to disrupt the microvillar cytoskeleton in order to analyze its possible role in immobilizing NHE3, using K depletion and Clostridium difficile toxin B (TxB), an agent that impairs Rho GTPase function and has profound effects on microvillar structure. The results provide evidence that detachment of the immobile fraction from its cytoskeletal anchorage leads to rapid internalization.

    For SEM, cells grown to confluence on 25-mm coverslips were fixed at 4°C with 4% paraformaldehyde for 30 minutes, washed, then labeled for 60 minutes with anti-HA antibody (1:500 dilution) in phosphate-buffered saline. Goat anti-mouse secondary antibody conjugated to 18 nm gold was then applied, followed by gold enhancement using GoldEnhance EM. Samples were dehydrated, mounted on stubs, coated with carbon, and then analyzed by using an XL30 environmental ESM (ESEM) (FEI). Images were generated through the combination of signals from the gaseous secondary electron detector and the backscatter electron detector. Analysis of particle distribution showed that 71% of the gold particles were associated with the apical membrane; however, treatment with TxB resulted in a drastic reduction in gold labeling, confirms that apical accumulation of NHE3 results, at least in part, from a retention process that involves Rho GTPases and the actin cytoskeleton.

    These observations suggest that modulation of the mobile fraction of NHE3 on the apical membrane can alter the number of functional exchangers on the cell surface and hence the rate of transepithelial ion transport. Regulation of the interaction of NHE3 with the actin cytoskeleton can therefore provide a new mode of regulation of sodium and hydrogen transport.

    Reference:

    Alexander, R. T.; Furuya, W.; Szaszi, K.; Orlowski, J., and Grinstein, S.: Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: role in apical retention and targeting. Proc. Natl. Acad. Sci. USA, 102, 12253-12258 (2005).

    Depending on the sample properties and experimental conditions, the rate of gold particle enlargement with GoldEnhance can vary, and in some systems very rapid development of larger particles has been reported. If you observe the rapid formation of larger particles after a few minutes, we recommend the following:

    1. Examine after a shorter development time. In some applications, 1-2 minutes may be enough to enlarge the particles to the desired size.

    2. Substitute Solution D of the GoldEnhance with 0.05M sodium phosphate with 0.1M sodium chloride, adjusted to pH 5.5. This will reduce the pH of the reaction mixture, which will serve to slow down the rate of the gold deposition reaction.

    3. Increase the sodium chloride concentration in your substitute for D to 0.5M. We have occasionally observed that higher ionic strengths reduces background, and may act to moderate the rate of development (possibly by shortening the range of ionic interactions in the solution).

    4. Add a viscosity modifier, such as polyethylene glycol (PEG). A solution of one of the higher molecular weight forms, such as 1% carbowax (molecular weight 20,000), is best; dissolve 4% in solution D or your substitute; gum arabic, although it is somewhat less convenient to prepare since it requires longer to dissolve, is also effective. These are used to slow down development in silver enhancement and lead to uniform particle size and morphology, and it is reasonable to expect the same result with gold enhancement.

    5. Adding a small amount of detergent, such as 0.1% Tween-20 (add as 0.4% in solution D or your substitute). Detergents are known to modify the growth of gold nanoparticles and may provide a growth constraint during gold enhancement.

    The most efficient strategy is to start with the first two suggestions; if these do not give the desired results, work through the other three, (3), (4) and (5) in the order given.

    More information:

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    MSDSs Now Available Online

    If you require a Material Safety Data Sheet (MSDS) for one of our products, you can now download them in PDF format from our web site. Links to MSDSs are located next to the links to product instructions in our online catalog pages near the top of the page, in the tables of contents in the HTML format product instructions, or on our new Materials Safety Data Sheet index page.

    In addition, the product information files in PDF format have been updated or replaced. All fonts are now embedded - so the display problems reported on some platforms with the previous versions have been fixed.

    More information:

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    Other Recent Publications

    Kollman and Quispe used our negative stain reagent, Nano-W to help solve the structure of the 420 kDa lobster clottable protein by single particle reconstruction. Crustaceans form clots by the rapid crosslinking of a hemolymph clottable protein (CP) to form long, branched polymers. Clotting limits hemolymph loss from wounds and plays a part in the innate immune response. CP is a 420 kDa homodimer with a large quantity of associated lipid, primarily the carotenoid pigment astaxanthin. The three-dimensional structure of CP from the lobster Panulirus interruptus has been determined to 17Å resolution by single particle reconstruction from electron micrographs of the protein embedded in vitreous ice. Three data sets were collected: a negative stain set, a high dose cryo data set from which an initial model was generated, and a large cryo reconstruction data set. For negative stain experiments, purified protein was diluted to 100 micrograms/mL in water, applied to 400 mesh copper grids with carbon support film and stained with Nano-W; for cryo-EM, purified protein was diluted to 1 mg/mL and applied to Quantifoil grids. The grids were blotted for 3.5 s and frozen using an automated vitrification device. The most prominent feature of this structure is a large cavity spanning the length of the molecule, which is the likely lipid binding pocket. The EM structure was used in a low resolution molecular replacement search with data from orthorhombic CP crystals: a solution is presented which describes the crystal packing.

    Reference:

    Kollman, J. M., and Quispe, J.: The 17Å structure of the 420 kDa lobster clottable protein by single particle reconstruction from cryoelectron micrographs. J. Struct. Biol., 151, 306-314 (2005).

    Our pre-embedding immunoelectron microscopy customers continue to go strong. Reporting in the current Journal of Histochemistry and Cytochemistry, Sakulsak and co-workers used this method to study the ultrastructural distribution of a novel 503 amino-acid protein named SLAMP (sublingual acinar membrane protein) obtained by cloning a rat gene and expressed primarily in the sublingual gland. SLAMP is a novel lectin with 63% homology with human ERGIC-53-like protein, in the family of animal L-type lectins. The expression and localization of SLAMP in major rat organs and tissues was examined using a cDNA probe for SLAMP mRNA and rabbit antisera against SLAMP; abundant expression of SLAMP was demonstrated predominantly in the sublingual gland, with single sizes of the mRNA and protein 1.8 kb and 50 kDa, respectively, but not in other organs or tissues, including the parotid and submandibular glands. Enzyme immunohistochemistry showed that SLAMP was localized to the mucous acinar cells, but not to the serous demilunes or the duct system. For electron microscopic immunocytochemistry, cryostat sections on plastic slides, after successive pretreatments with 0.3% Tween 20 and 3% normal swine serum, were incubated with anti-SLAMP antisera at 1:1000 dilution overnight at room temperature, washed in phosphate-buffered saline (PBS) and incubated with Nanogold®-Fab' goat anti-rabbit at 1:100 dilution in PBS plus 0.5% BSA (PBS-BSA) for 1 hour. After washing with PBS-BSA, the sections were postfixed in 1% glutaraldehyde in PBS for 10 min, washed thoroughly in distilled water (DW) and developed with HQ silver for 5 minutes in the dark room. They were then washed in DW, postfixed with 0.5% osmium tetroxide for 15 minutes, washed and dipped in DW overnight at 4°C, then stained with 2% uranyl acetate. Finally, they were dehydrated in ethanol series and embedded in an epoxy resin. Ultrathin sections were made and examined in the electron microscope. SLAMP was localized predominantly to regions corresponding to the ER-Golgi intermediate compartment. Besides the sublingual gland, SLAMP immunoreactivity was also demonstrated in mucous cells of the minor salivary glands in oral cavity and of Brunners glands in the duodenum, suggesting that rat SLAMP plays a specific role in the early secretory pathway of glycoproteins in specific types of mucous cells.

    Reference:

    Sakulsak, N.; Wakayama, T.; Hipkaeo, W.; Yamamoto, M., and Iseki, S.: Cloning and characterization of a novel animal lectin expressed in the rat sublingual gland. J. Histochem. Cytochem., 53, 1335-1343 (2005).

    Kloc and co-workers describe the use of a Nanogold-labeled anti-digoxigenin antibody, prepared by Nanogold labeling a comercially available antibody, for RNA in situ hybridization in their recent paper in Development. Light and electron microscopy of whole-mount in situ hybridization specimens. Organization of cytokeratin filaments, but not the actin cytoskeleton, depends on the presence of intact VegT mRNA and a noncoding RNA, Xlsirts. Destruction of either of these transcripts results in disruption of the cytokeratin cytoskeleton in a transcript-specific manner and interferes with proper formation of the germinal granules and subsequent germline development. In addition, analysis of the distribution of endogenous VegT and Xlsirts in live oocytes using molecular beacons showed that these RNAs are integrated into the cytokeratin cytoskeleton. These results demonstrate a novel structural role of coding and noncoding RNAs in the organization of the vegetal cortex of Xenopus oocytes.

    Reference:

    Kloc, M.; Wilk, K.; Vargas, D.; Shirato, Y.; Bilinski, S., and Etkin, L. D.; Potential structural role of non-coding and coding RNAs in the organization of the cytoskeleton at the vegetal cortex of Xenopus oocytes. Development, 132, 3445-3457 (2005).

    Details of preparation and method:

    Kloc, M.; Bilinski, S.; Chan, A., and Etkin, L. D.: Mitochondrial ribosomal RNA. in the germinal granules in Xenopus embryos-revisited. Differentiation, 67, 8083 (2001).

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