N A N O P R O B E S E - N E W S
Vol. 8, No. 10 October 31, 2007
Updated: October 31, 2007
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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Because of their small size, antibody fragments labeled with Nanogold or Undecagold provide an ideal combination of features for imaging the 3-dimensional distribution of proteins or other targets in cells by electron tomography:
- High penetration into cells and tissues.
- High labeling density.
- Close to quantitative labeling of antigenic sites.
- High labeling resolution.
The only problem is that their small size can make them difficult to detect by conventional bright-field electron tomography. However, in their recent paper in the Journal of Structural Biology, Sousa, Leapman and co-workers use Scanning Transmission Electron Microscopy (STEM) to determine the detection limits and optimal experimental conditions for imaging these smaller particles, and their conclusions will be useful for anyone contemplating using these probes in applications such as tomography or thick sections where resolution is a concern.
Resolution advantage: size comparison of Nanogold-Fab' with conventional 5 nm colloidal gold-IgG probe, showing overall probe size and distance of gold from target. Due to its position at the hinge region, Nanogold is positioned closer to the target upon binding, yet does not hinder or interfere with binding.
Stability of the gold clusters under electron irradiation was tested using preparations of undecagold or Nanogold embedded in carbon films. Ultrathin carbon films were prepared by evaporating approximately 3 nm of carbon onto fresh cleaved mica, floated in water, and collected on 200-mesh grids covered with lacey carbon film. The grids were glow-discharged in air to facilitate adsorption of the gold clusters, and 5-ll droplets of 1:50 diluted Nanogold or 5-ll droplets of 1:20 diluted Undecagold were deposited onto the surface of the grids. After 2 minutes adsorption, the grids were blotted with filter paper, then washed five times with 5 µL aliquots of deionized water. An additional thin (approximately 1 nm) carbon layer was deposited onto some of the grids to investigate whether this improved the stability of the gold clusters in the electron beam.
A specimen containing Nanogold embedded in a 100 nm thick layer of carbon was prepared by depositing Nanogold onto an approximately 35 nm evaporated carbon layer supported on a lacey grid. An additional 65 nm layer of carbon was then evaporated onto the specimen on the same side as the deposited Nanogold, giving a total specimen thickness of 100 nm, with the nanoparticles sandwiched as a layer within a carbon matrix. Larger, 10 nm diameter colloidal gold particles were then deposited onto the top and bottom of the surfaces to serve as fiducials for drift correction during acquisition of the tilt series and for image alignment before 3D reconstruction. Specimens containing Undecagold embedded in a carbon film were prepared using the same procedure, except that the first layer of evaporated carbon was approximately 15 nm in thickness and the second layer 5 nm, with 3 nm-diameter colloidal gold particles as fiducials.
To assess the visibility of Nanogold in a lightly stained cell, an 80 nm-thick section of plastic-embedded Caenorhabditis elegans was prepared. The C. elegans were placed in a cryopreservative solution of 15% sucrose and frozen in a high-pressure freezing machine. The frozen blocks were then processed at low temperature in a freeze-substitution system using a solution of acetone containing 1.0% osmium tetroxide. After freeze-substitution for 3 days, specimens were gradually warmed to room temperature and embedded in EponAraldite by graded exchange of the acetone, then polymerized in 100% resin by heating to 60°C for two days, sectioned to a nominal thickness of 80 nm with an ultramicrotome, and mounted on 300 mesh EM copper grids. Both Nanogold and 10 nm colloidal gold were deposited on top of the sections to test the visibility of the Nanogold in the presence of light stain, with the larger particles serving as fiducials in the alignment of the tilt series.
Acquisition of tomographic tilt series was performed on a 300 kV field-emission microscope (FEI) with a HAADF detector situated after the projection-lens system above the viewing screen. FEI software was used to acquire the tomographic tilt series. STEM tomography of the specimen of Nanogold embedded in carbon was acquired with a tilt range of -60° to +60° in steps of 3°, with an integral dose of 105 e/nm2, and a pixel size in the STEM image of 0.37 nm. The FWHM of the STEM probe was measured on a slow-scan CCD camera and determined to be about 0.6 nm. During data acquisition, the probe was always focused on the surface of the specimen. The inner collection semi-angle of the ADF detector was 16 mrad, and the outer semi-angle five times the inner angle (80 mrad), with convergence semi-angle of the beam on the specimen of 10 mrad. For the Undecagold embedded in carbon, STEM tomography was conducted with an integral dose of 3.5 x 105 e/nm2, a pixel size of 0.23 nm, and a tilt range from -60° to +60° in steps of 2°.
The STEM tomographic tomographic tilt series of C. elegans was acquired with a tilt range from -60° to +60° in steps of 2°, with a total dose of 2 x 105 e/nm2, and with a pixel size of 0.3 nm. Images were 2k x 2k pixels in for all tomographic series. For 3D reconstruction, the images at each tilt increment were aligned in the IMOD software using fiducial markers. In some cases, features corresponding to the gold fiducial markers were removed after alignment of the tilt series and prior to reconstruction to eliminate artifactual streaks in the final tomogram caused by the limited tilt range of the dataset. Tomographic reconstruction of the aligned tilt series was performed using the simultaneous iterative reconstruction technique (SIRT) algorithm. The specimen
of Nanogold embedded in 100 nm of carbon was also reconstructed with weighted back-projection (WBP).
To assess the visibility of the gold clusters in ADF STEM images, the elastic scattering from Undecagold clusters embedded in a thin layer of amorphous carbon was modeled by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. For simplicity, only the 11 gold atoms in the cluster were considered. Scattering was assumed to be incoherent and the specimen sufficiently thin that only single elastic scattering events have to be considered. The simulations indicated that visibility in 2 dimensions of undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (1520 mrad). Furthermore, the visibility of Undecagold in 3-D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density.
The beam damage response of the two labels was evaluated at an acceleration voltage of 300 kV. The decrease in total integrated signal within Nanogold and Undecagold particles deposited onto ~3 nm-thick carbon films as a function of dose showed that Nanogold lost only 5% of its mass over a dose range of 2.0 x 104 e/nm2 to 4.5 x 105 e/nm2, while undecagold showed 35% mass loss under the same dose conditions. However, the damage response curve for Undecagold clusters coated with approximately 1 nm of carbon indicated that even such thin layers of carbon are enough to decrease the total mass loss for undecagold from 37% to 19%. Therefore, despite its sensitivity to incident electron irradiation when deposited on the surface, undecagold should be useful for immunolabeling - as long as the clusters are contained within a plastic section.
The simulations indicated that undecagold could be detected within 40 nm specimens. Experimental measurements successfully located undecagold clusters within 20 nm carbon sections, but not within 50 nm carbon sections: this was attributed to specimen drift and contamination build-up. Taking into account the difference in density between evaporated carbon (2 g/cm3) and Epon resin (1.25 g/cm3), it should therefore be feasible to detect Undecagold clusters in 64 nm-thick plastic sections, a sufficient thickness to yield relevant 3D structural information within sectioned cells. Measurements with Nanogold resulted in clear imaging within 100 nm carbon sections, and demonstrate that it is possible to detect Nanogold particles in 80 nm plastic sections of tissue freeze-substituted in the presence of osmium. These results indicate that STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with undecagold and Nanogold, and this quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these cluster labels.
- Sousa, A. A.; Aronova, M. A.; Kim, Y. C.; Dorward, L. M.; Zhang, G, and Leapman, R. D.: On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography. J. Struct. Biol., 159, 507-522 (2007).
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If you have been using Nanogold®: for detection on nitrocellulose membrane blots (immunodot blots or Westerns) and experienced less than ideal detection, we have developed an optimized detection procedure that maintains the already very high sensitivity, but combines it with a greatly reduced background and enhanced signal clarity. For the best results, we recommend using a procedure that incorporates the following features:
- Use gold enhancement rather than silver enhancement to develop the signal after application of the Nanogold conjugate. In most cases, this will provide substantially lower background or non-specific signal, while maintaining a similar level of sensitivity to silver enhancement.
- Incorporate 0.1% Tween-20 (detergent) in the buffers used for blocking, antibody incubation, and washing. This will dramatically reduce background binding.
- Include 1% nonfat dried milk (you can use the material sold in supermarkets and food stores) as an additive in the incubation buffer (the buffer in which the Nanogold is dissolved and applied to the blot) and 5% nonfat dried milk in the blocking buffer used to block the membrane before application of antibodies.
REAGENTS AND EQUIPMENT:
- Phosphate buffered saline (PBS): 20 mM sodium phosphate buffer pH 7.4 and 150 mM NaCl.
- Specific antigen (target protein or other biomolecule).
- Nitrocellulose (NC) membrane 0.2 µm pore size.
- Blotting Paper to wick membrane dry.
- Orbital Shaker
- Washing buffer (TBS-Tween 20): 20 mM Tris pH 7.6, with 150 mM NaCl and 0.1 % Tween-20.
- Nonfat dried milk (Carnation)
- GoldEnhance EM (Nanoprobes Product No. 2113).
- Specific Nanogold antibody conjugate.
- Prepare antigen solutions with a series of dilutions (0.01mg/mL, 0.001mg/mL, 0.0005mg/mL, 0.0001 mg/mL, 0.00005 mg/mL, 0.00001 mg/mL and 0.000005 mg/mL) using PBS, pH7.4.
- Pipette 1 µL of above solutions to a dry nitrocellulose membrane; prepare 2 duplicates as a negative control.
- Negative control 1: No antigen, No antibody.
- Negative control 2: No antigen with NG-conjugate incubation.
- Air-dry for 30 minutes
- Immerse membranes in 8 mL of TBS-Tween 20 for 5 minutes.
- Block membranes in 8 mL of TBS-Tween 20 containing 5 % nonfat dried milk for 30 minutes at room temperature.
Binding of Nanogold antibody conjugate:
- Dilute Nanogold antibody conjugate in TBS-Tween20 containing 1% nonfat dried milk to 4 µg/mL (1:20 Dilution: 300 µL conjugate + 5.30 ml TBS-gelatin containing 1% nonfat dried milk).
- Incubate the membranes in 8 mL of diluted conjugate solution for 30 minutes at room temperature.
- Incubate the control membrane in 8 mL of TBS-Tween20 containing 1% nonfat dried milk for 30 minutes at room temperature.
- Wash membranes three times for 3 min each in 8 mL of TBS-Tween 20. Wash membranes thoroughly in 8 mL of deionized water (4 x 3 minutes). Make sure strips are washed separately according to what they are incubated in (strips incubated in one lot of a conjugate are washed in a separate dish from strips that are incubated in TBS-Tween 20 with 1% nonfat dried milk without conjugate, strips incubated in different lots are washed separately).
- Perform Gold Enhancement according to instructions (mix solutions A and B, wait 5 minutes, then add C and D).
- Record the number of observed spots and time when the spots appear. Record the time when background appears on the control membrane.
- After 15 minutes, the enhancement solution is removed. Rinse membranes with water (3 x 3 minutes) and air-dry for storage.
Tween-20 and nonfat dried milk should also improve the performance of Nanogold conjugates used with silver enhancement.
Examples of detection with Nanogold on blots are shown below, in comparison with detection using conventional colloidal gold.
Blot test with Nanogold and colloidal gold conjugates. (a) dot blot image of Nanogold-Fab' Goat anti-Rabbit IgG, developed with GoldEnhance EM gold enhancement reagent, using the Schleicher & Schuell (recently acquired by Whatman) MINIFOLD-1 Dot Blot System which applies the target precisely over a circle with a diameter of 5 mm. The 5 mm circle is big enough for a quantitative analysis by a densitometer. With the target spreading over 5mm circle, we can detect as low as 0.01 ng antigen. While we use a pipette to apply 1 L of target over a circle of 1-2 mm, we can detect as little as 0.005 ng by the same NG conjugates. (b) Nanogold anti-mouse Fab' blotted against mouse IgG, developed with LI Silver (Nanoprobes), showing sensitivity enhancement with smaller spot size. Target was applied using a 1 µL microcapillary tube. This immunodot blot shows 0.1 pg sensitivity (arrow). (c) Colloidal gold blot using a 15 nm colloidal gold conjugate to detect serial dilutions of an IgG target, without silver or gold enhancement. The range of color densities should give a good basis for comparing your results - i.e. if your spot is darker than our 50 ng spot your conjugate is working very well, but if it is much fainter, it is questionable.
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Gold enhancement is an autometallographic method, similar to silver enhancement, but in which gold rather than silver 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 any strength osmium tetroxide - it is not etched.
- May be used in physiological buffers (including chlorides, which 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 for SEM observation.
- GoldEnhance is near neutral pH for best ultrastructural preservation.
- Low viscosity, so the components may be dispensed and mixed easily and accurately.
GoldEnhance represents another approach to visualizing Nanogold in situations where it is too small to be readily resolved. In their current paper in the Journal of Structural Biology, Wanzhong He and the group of Dr. Pamela J. Bjorkman of the Division of Biology at Caltech describe its use with endocytosed Nanogold probes for the study of dynamic events during endocytosis and subsequent intracellular trafficking by high pressure freezing/freeze substitution fixation (HPF/FSF). This is the most accurate method for preserving ultrastructure; however, its use with Nanogold was previously limited because the small size of the Nanogold prevented visualization.
The authors evaluated methods to enlarge 1.4 nm Nanogold labels bound to IgG Fc directed against the neonatal Fc receptor (FcRn). FcRn displays a strongly pH-dependent binding affinity transition: FcRn binds ligand at the acidic pH found in the gut and intracellular endosomes (pH 6 to 6.5) but not at the pH of blood (pH 7.4). This behavior is critical for FcRns functions in transporting maternal IgG across epithelial cell barriers, providing immunity to offspring, and in rescuing serum IgG from degradation. Fc fragment from rat IgG2a was expressed in Chinese hamster ovary cells; optimum conditions to selectively reduce hinge disulfides were obtained by evaluating the results of different reduction protocols using Ellmans Reagent. 1.01.5 mg of Fc was reduced with 1218 mg of mercaptoethylamine hydrochloride (MEA) in 1 mL of 0.1 M NaPO4, pH 6.0, 5 mM EDTA for 1.5 hours at 37°C, then passed over a Superdex-75 size exclusion column in 20 mM NaPO4, pH 6.5 with 150 mM NaCl and 1 mM EDTA. After concentration, the reduced Fc was labeled with Monomaleimido Nanogold, which reacts specifically with reduced sulfhydryls: ~30 nmol of Monomaleimido Nanogold was suspended in 1 mL deionized water, then immediately incubated with the reduced Fc for 24 hours at room temperature. Labeled Fc (AuFc) was separated from unlabeled Fc and free Nanogold by gel filtration using the Superdex 75 column. Labeled Fc was further purified by passage over an FcRn affinity column at pH 6, followed by elution at pH 8. Molar concentrations of Nanogold and Fc were determined spectrophotometrically using absorptions at 420 and 280 nm respectively, using extinction coefficients of 155,000 M-1cm1 for Nanogold and 60,900 M-1cm1 for Fc.
Labeled Fc uptake was investigated using 11 - to 13 - day suckling rats born to SpragueDawley rats. These were separated from their mothers for ~3 hours, then fed 3 x 100 µL (at 4560 minute intervals) of AuFc (~2 µM) in 20 mM NaPO4, 1.2 mM CaCl2, 0.5 mM MgCl2, 0.25 mM MgSO4, pH 6.0 at 37°C. After 23 hours, the rats were anesthetized with CO2, sacrificed, and the small intestine removed by dissection. A 45 cm segment of proximal small intestine, starting ~2 cm from the pylorus, and a 45 cm segment of the distal small intestine (ileum), ~2 cm from the ileocecal valve, were removed for chemical fixation or high pressure freezing. Small intestine segments were quickly cut into small pieces and immediately fixed in 3% glutaraldehyde / 1% formaldehyde in 0.1 M sodium phosphate, pH 6.0, 2 mM CaCl2, 1mM MgCl2, 0.5 mM MgSO4 for 30 minutes at 20°C, then a further 34 hours at 4°C in the same buffer at pH 7.4, then rinsed with 0.1 M sodium phosphate, pH 7.4, before gold enhancing.
For the gold enhancement step, the authors required consistent particle size and a slower and more controlled rate of development. Therefore, they developed an improved gold enhancement protocol:
- Wash samples 3 x 5 minutes with PBS including glycine (20 mM sodium phosphate, pH 7.4, 150 mM
NaCl, 50 mM glycine) to remove aldehydes.
- Wash samples 3 x 5 minutes with PBSBSA (bovine serum albumin)Tween (PBS containing 1% BSA and
0.05% Tween 20)
- Wash samples 3 x 5 minutes with 5 mM sodium phosphate, pH 5.5, 100 mM NaCl ("Solution E").
- For gold enhancement, place 15 samples (~1 mm x ~1 mm) in a mixture of GoldEnhance EM Solutions A and B at a 2 : 1 ratio (~80 µL of A and ~40 µL of B). After 5 minutes, added ~200 µL of "Solution E" with 20% gum arabic, then ~80 µL of Solution C.
- After 7 15 minutes of development, transfer samples to 12% sodium thiosulfate to stop enhancement.
- Wash 3 x 5 minutes with buffer E, then rinse with water.
This protocol resulted in slower development, reduced background and improved size uniformity of the enhanced particles, especially when the enhancement was conducted at 4°C rather than room temperature. If the gum arabic was omitted, size variations were observed in the enlarged gold particles, perhaps resulting from differences in the time it took for the sodium thiosulfate 'stop' solution to reach different portions of the cell. Gold-enhanced, chemically fixed samples were incubated in 1% OsO4 in 0.1 M sodium phosphate, pH 6.1, for 60 minutes, rinsed with distilled water, then stained 1 hour en bloc with 2% uranyl acetate. The samples were dehydrated with progressive lowering of temperature.
Intestinal samples were rapidly frozen with a High Pressure Freezing Machine. An intestinal segment was transferred into the 200 µm deep side of a 100 µm/200 µm specimen carrier that was ~2 mm in diameter. The specimen chamber was filled with 1-hexadecene, then sandwiched against the flat side of a 300 µm specimen carrier. The sandwiched carrier was placed in the sample holder, then high pressure frozen at 2100 bar and transferred to liquid nitrogen for storage. Time from initial cutting of the sample and freezing was 3040s. For conventional FSF of unenhanced samples, the specimen carriers with frozen samples were transferred to 1.5 mL microcentrifuge tubes (Fisher Scientific, USA) containing a frozen solution of acetone with 1% OsO4 and 0.1% uranyl acetate under liquid nitrogen. Tubes were placed in a Leica EM AFS machine (Leica Microsystems) at -140°C and gradually warmed to -90°C in 4 hours. The temperature was then gradually raised in 6 h transitions in the Leica AFS system as follows: -90°C for 2448 h, -60°C for 24 h, and -30°C for 18 h. After slowly warming to 0°C over 2 hours, samples were washed three times in pure acetone and warmed to room temperature.
Chemically fixed or HPF/FSF intestinal samples were infiltrated, embedded, polymerized, sectioned and stained as previously described (He et al., 2003). Selected regions of 70200 nm sections were examined using an Electron Microscope operating at 120 kV, and projection images recorded on a 2K x 2K CCD camera. Tilt series (70°, 1.0° angular increments) were digitally recorded at 6500 x (pixel size = 1.57 nm) and at 700 nm underfocus about two orthogonal axes, using the microscope control program SerialEM. Tomograms were computed for each tilt axis using the enlarged gold particles as markers for alignment, then aligned to each other and combined using the IMOD software package.
Initial results using gold-toned silver-enhanced particles demonstrated that AuFc retains pH-dependent binding to FcRn, is monodisperse, and is endocytosed normally into FcRn-expressing cells: the gold does not affect endocytosis. The conjugates were then used to optimize the pre-embedding gold enlarging techniques described above. The optimized chemical fixation enhancement method resulted in reduced autonucleation, and our HPF/FSF method demonstrated that gold enhancement can be used to gradually enlarge Nanogold and render it impervious to osmium compounds used during fixation. The resulting heavy metal labels were large enough to be visible in 2D projections and tomograms, yet small enough to provide a spatial resolution about as good as EM tomography itself. This allowed the reliable identification of individual FcRn ligands inside intracellular vesicles in cells expressing FcRn. These methods should be applicable to a broad range of endocytosis and transcytosis studies.
In some situations, GoldEnhance may produce a fine, "granular" non-specific background signal of small particles. There are several ways in which you can either slow down development so that you have more control over its progress, or chemically "stop" the development process. Which approach is best for the system under study depends upon whether you believe the primary problem is continued development after rinsing, in which case a chemical "stop" is appropriate, or excessively fast development before rinsing, in which case slowing down the reaction is most appropriate. Fine background that seems to arise from continued development after rinsing to remove the GoldEnhance reagents may be removed using a number of "stop" procedures: the simplest and most universal is treatment with 1% or 2% freshly prepared sodium thiosulfate for a few seconds, and unless there are specific reasons that this would be harmful to your specimen, we usually suggest that you try this first. Another "stop" treatment, described previously by Wanzhong He, is treatment with 3% Na2S2O3 for 30 seconds, then washing with 10% acetic acid + 10% glucose for 3 x 5 minutes. We have discussed alternative strategies for controlling background more extensively on our web site.
Some preliminary images are shown below:
left: Gold enhancement of intestinal tissue with Nanogold staining. (a) Nanogold particles, enhanced with GoldEnhance EM for 3 minutes; (b) control with Nanogold labeling omitted, showing the granular background. right: analogous procedure, using modified protocol to control background, comprising: one part A (enhancer) : 5 parts B (activator); D (buffer) was substituted with 0.05M sodium phosphate with 0.1M sodium chloride, at pH 5.5; enhancement was performed for 10-15 minutes rather than 3 minutes, "stopped" using 3% Na2S2O3 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes. (c) Nanogold particles, enhanced with GoldEnhance EM; (d) control with Nanogold labeling omitted, identical gold enhancement procedure. Insets show experimental (left) and control (right) specimens (Thanks to Wanzhong He and Dr. Pamela Bjorkman for the images).
- He, W.; Kivork, C.; Machinani, S.; Morphew, M. K.; Gail, A. M.; Tesar, D. B.; Tiangco, N. E.; McIntosh, J. R., and Bjorkman, P. J.: A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules. J. Struct. Biol., 160, 103-113 (2007).
Reference for sample preparation for HPF/FSF:
- He, W.; Cowin, P., and Stokes, D. L.: Untangling desmosomal knots with electron tomography. Science, 302, 109113 (2003).
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Fras1 is the first identified member of a family of extracellular matrix proteins family that also includes Frem1, Frem2 and Frem3. In mouse, mutations in Fras1, Frem1 and Frem2 have been associated with the bleb phenotype, while loss of Fras1 function in humans is causative for Fraser syndrome. Common phenotypic features between Fras1-/- embryos and Fraser syndrome patients include formation of embryonic sub-epidermal blisters, unilateral or bilateral renal agenesis or dysgenesis, cutaneous syndactyly and cryptophthalmos (covered eyelids); in addition, Fraser syndrome is accompanied by other developmental defects, including genital malformations, heart, airway and lung anomalies and abnormalities.
Dalazios and co-workers, in their recent paper in the Archives of Dermatological Research, use the resolution and penetration of Nanogold-labeled Fab' fragments to study the ultrastructural distribution of Fras1 and its relation to bleb symptom localization. Immunofluorescence showed that Fras1 co-localizes with the markers of epithelial basement membranes, consistent with its effects on the cohesiveness of the embryonic skin basement membrane with its underlying mesenchyme. The ultrastructural localization pattern of Fras1 was compared in two epithelial basement membranes: the skin epidermis, which is largely affected by the bleb phenotype, and the esophagus, which histologically appears normal in Fras1-/- mutants. At the level of electron microscopy, epithelial basement membranes are divided into two distinct layers, according to their electron density: the lamina lucida, an electron-lucent layer which contains anchoring filaments extending from the hemidesmosomes to a more electron-dense layer referred to as the lamina densa. A third layer, lamina fibroreticularis or sublamina densa, forms the link between the lamina densa and the underlying connective tissue, which is maintained by anchoring fibrils mainly consisting of collagen type VII.
Pre-embedding Nanogold labeling was used to localize Fras1 in mouse embryos. Mouse E14.5 embryos (from NMRI strain) were dissected out of the uterus and fixed overnight in 4% paraformaldehyde (PFA) with 0.05% glutaraldehyde and 15% saturated picric acid in 0.1 M phosphate buffer, pH 7.2, then embedded in gelatin and sectioned at 80 µm with a vibrating microtome. Free floating sections were pre-incubated in 20% normal goat serum (NGS) diluted in TBS-buffer (50 mM Tris, 0.9% NaCl, pH 7.4), for 1 hour, and then incubated for 48 hours at 4C with the primary antibody (1:30) diluted in 1% NGS in Tris-buffered saline (TBS). After several washes in TBS, sections were incubated with Nanogold-labeled goat anti-rabbit affinity purified Fab fragments. After washing with TBS followed by several rinses in double-distilled water, the gold particles were silver enhanced for 812 minutes using the HQ Silver kit. Sections were then treated with 1% OsO4 and contrasted in 1% uranyl-acetate before dehydration and embedding in epoxy resin. Serial electron microscope sections (7080 nm) were collected on pioloform-coated copper slot grids. Images were obtained using a Transmission Electron Microscope operating at 80 kV.
Fras1 immunoreactivity was detected in all epithelia examined. The protein was localized both in the dermalepidermal junction, as well as in the basement membrane of esophagus, displaying a clustered deposition almost exclusively below the lamina densa. Extensive deposition of gold particles was not observed within the lamina densa or the lamina lucida, indicating that at least the central NG2-like domain of Fras1 is located within the lamina fibroreticularis or sub-lamina densa. These results do not exclude the possibility that other domains of Fras1, which is a large 4,010 - amino acid protein) are located within either the lamina densa or the lamina lucida. Clustered gold/silver enhanced depositions were observed, usually attached to anchoring fibrils. Clusters corresponding to Fras1 were frequently detected in close proximity to mesenchymal cells, indicating that Fras1 could serve as a direct link between the sublamina densa and mesenchyme; Fras1 also contains a putative cell recognition RGD sequence, which could serve for the attachment of Fras1 to mesenchymal cells, but this assumption requires experimental confirmation. The localization of Fras1 is consistent with previous results indicating that Fras1 exerts its function below the lamina densa, and that Fras1 displays the same localization pattern in all epithelial basement membranes.
- Dalezios, Y.; Papasozomenos, B.; Petrou, P., and Chalepakis, G.: Ultrastructural localization of Fras1 in the sublamina densa of embryonic epithelial basement membranes. Arch. Dermatol. Res., 299, 337-343 (2007).
Additional method details:
- Dalezios, Y.; Lujan, R.; Shigemoto, R.; Roberts, J. D, and Somogyi, P.: Enrichment of mGluR7a in the presynaptic active zones of GABAergic and non-GABAergic terminals on interneurons in the rat somatosensory cortex. Cereb Cortex., 12, 961-974 (2002).
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We have some exciting new products coming down the pipeline tailored for blotting and histochemical staining. We will be announcing these at the Annual Meeting of the American Society for Cell Biology in Washington, DC on December 1-5 this year. We will be located in booth 1241.
We will also be presenting two papers at the meeting describing some of our new technology. Both will be presented as posters:
- In the afternoon poster session 231, "New and Emerging Technologies for Cell Biology II," on Monday, December 3, we will be presenting new results with our enzyme metallography (EnzMet™) technology.
- In session 326, "Molecular Biology and Detection," on the afternoon of Tuesday, December 4, we will present Rapid detection of His-tagged proteins on Western Blots with GoldiBlot™
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Surface Enhanced Raman Spectroscopy (SERS) is a highly sensitive method for studying the vibrational spectra of organic or biological molecules. When these molecules are adsorbed onto gold or especially silver nanoparticles, Raman spectral bands for some molecules are greatly intensified, sufficiently to provide a sensitive method for the detection of these molecules. In a recent issue of the Journal of the American Chemical Society, Braun and group describe a label-free sensing platform using SERS for the unambiguous detection of single-stranded DNA. A 24-mer oligonucleotide was used as a model target strand. Silver nanoparticles (~15 nm diameter) were functionalized with the complement of one-half of the target strand; a stable and smooth SERS-inactive silver film (thickness ~100 nm) was modified with the complement of the remaining portion of the target sequence. The silver film was then treated with 6-mercaptohexanol (MCH) to prevent nonspecific binding of DNA, nanoparticles, or impurities to its surface, and to improve hybridization efficiency. The film was then labeled with 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino) fluorescein (SAMSA fluorescein), which contains a protected thiol, as a surface-bound Raman label; this absorbs close to the laser excitation wavelength of 514.5 nm. The labeled Film was incubated with the target strand and the complementary silver nanoparticle solution, consecutively, capturing in turn the targets and the functionalized nanoparticles through sequence-selective hybridization. After washing to remove non-specifically bound NPs, the hybridized surface was dried in a nitrogen stream, and Raman measurements were carried out under ambient conditions. AFM imaging showed that only a few nanoparticles (and therefore fewer than 100 Raman label molecules) are sufficient to give rise to intense and reliable SERS signals, making this a potentially useful biosensing method.
- Braun, G.; Lee, S. J.; Dante, M.; Nguyen, T. Q.; Moskovits, M., and Reich, N.: Surface-enhanced Raman spectroscopy for DNA detection by nanoparticle assembly onto smooth metal films. J. Amer. Chem. Soc., 129, 6378-6379 (2007).
New references describing the use of Nanogold for pre-embedding labeling come along fairly frequently, and in addition to the excellent results of Dalezios mentioned above, Askari and colleagues present more results with this application in a recent paper in Virchows Archiv where they used it for pre-embedding localization of globotriaosylceramide, the main glycolipid that accumulates in Fabry disease. Skin biopsy or cell culture material was fixed in freshly made 4% paraformaldehyde in phosphate-buffered saline (PBS) for 112 hours. Fixed biopsies were vibratome sectioned to give 100 µm thick slices, fixed with 4% glutaraldehyde in 0.1 N sodium cacodylate buffer overnight for electron microscopic examination or processed for pre-embedding immunogold labeling. Vibratomed slices were washed in PBS, blocked, permeabilized with saponin (0.1% in PBS with 5% normal goat serum) for 1 hour, treated with mouse anti-Gb3 monoclonal antibody (1:400) for 12 hours, washed, then treated with Nanogold-labeled secondary antibody (1:250) for 1 hour, and enhanced for 1020 minutes with HQ Silver from Nanoprobes. Slices were then treated with 0.2% OsO4 in phosphate buffer for 30 minutes, mordanted en bloc with 0.25% uranyl acetate overnight at 4°C, dehydrated with ethanol, flat embedded in epoxy resin, and finally counterstained with uranyl acetate and lead citrate. Both the Nanogold pre-embedding labeling and postembedding immunogold electron microscopy of skin biopsies and untreated patient cultured skin fibroblasts using 15 nm gold confirmed the presence of globotriaosylceramide in the cell membrane, in various cytoplasmic structures, and in the nucleus. Control organ tissues and cultured fibroblasts from five unaffected subjects were all negative for globotriaosylceramide by immunohistochemistry and immunoelectron microscopy. A substantial amount of lysosomal and extralysosomal globotriaosylceramide immunoreactivity was found in cells and tissues even after years of enzyme replacement therapy in Fabry disease. In addition to enabling improved understanding of the disease mechanism, this suggests that immunostaining for globotriaosylceramide is a potentially useful means to assess response to novel therapies. While we are impressed with these results, we would like to point out that Nanogold is actually 1.4 nm in size, not 5 nm as stated in the results.
- Askari, H.; Kaneski, C. R.; Semino-Mora, C.; Desai, P.; Ang, A.; Kleiner, D. E.; Perlee, L. T.; Quezado, M.; Spollen, L. E.; Wustman, B. A., and Schiffmann, R.: Cellular and tissue localization of globotriaosylceramide in Fabry disease. Virchows Arch., 451, 823-834 (2007).
More details of method:
- Tao-Cheng, J. H.; Vinade, L.; Smith, C.; Winters, C. A.; Ward, R.; Brightman, M. W.; Reese, T. S, and Dosemeci, A.: Sustained elevation of calcium induces Ca(2+)/calmodulin-dependent protein kinase II clusters in hippocampal neurons. Neuroscience, 106, 6978 (2001).
Meanwhile, Bhattacharya and group report a simple one-step method of attaching folic acid (FA) to gold nanoparticles (AuNPs) for use as a targeted cancer imaging agent, and its fine tuning using different polyethylene glycol (PEG) backbones. 5 nm gold particles were prepared by borohydride reduction of a tetrachloroaurate solution, then adsorbed to amino-modified polyethylene glycol (PEG) of various lengths followed by folic acid (FA). PEG-diamine backbones with molecular weights 2000 (PAM2-2K) and 10,000 (PAM2-10K) and PEG-tetramine with molecular weight 20,000 (PAM4-20K), and PEG-dithiol with molecular weight 2000 (PSH2-2K) were compared, and the nanoconjugates characterized with ultraviolet-visible spectroscopy, transmission electron microscopy (TEM), thermogravimetric analysis, Fourier transform infrared spectroscopy, inductively coupled plasma analysis, and radioactivity measurements using 3H labeled folic acid to determine attachment and release profiles from the nanoconjugates. Folic acid binding was found to follow the order Au-PAM4-20K > Au-PAM2-10K > Au-PAM2-2K > Au > Au-PSH2-2K, and its release profile follows the reverse order. Therefore, Au-PAM4-20K-folic acid was used for folate receptor (FR)mediated targeting of AuNPs to cancer cells. Seven cancer cell lines (SKOV-3, OVCAR-5, OV-202, OV-167, OPM-1, RPMI, and U266) were screened for expression of FRs: among ovarian cancer cells, the expression pattern of FRs follows the order OV-167 > SKOV-3 > OV-202 > OVCAR-5, and multiple myeloma cell lines follow the order OPM-1 > U266 > RPMI. Intracellular uptake of nanoconjugates containing folic acid or lacking folic acid were monitored with digital optical photography and TEM. Quantitation of uptake was determined by gold analysis with inductively coupled plasma emission spectrometry. Uptake of the folic acid-gold nanoconjugates was found to correlate with FR expression: maximum uptake was observed for OV-167, minimum for OVCAR-5. TEM images of cells treated with Au-PAM4-20K-FA confirm the endocytosis of the nanoconjugates. This study provides important confirmation of the potential for targeted delivery of anticancer drugs, as well as metal nanoparticles for targeted therapy, tumor imaging, and ablation using overexpression of FRs on cancer cells.
- Bhattacharya, R.; Ranjan Patra, C.; Earl, A.; Wang, S.; Katarya, A.; Lu, L.; Kizhakkedathu, J. N.; Yaszemski, M. J.; Greipp, P. R.; Mukhopadhyay, D., and Mukherjee, P.: Attaching folic acid on gold nanoparticles using noncovalent interaction via different polyethylene glycol backbones and targeting of cancer cells. Nanomedicine, 3, 224-238 (2007).
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