N A N O P R O B E S     E - N E W S

Mice were sacrificed by CO2 narcosis and tissues were immediately fixed in 4% paraformaldehyde in 0.02 M PBS, pH 7.4, for 4 hr at room temperature (RT), washed in 0.02 M PBS, pH 7.4, and immersed overnight in 4°C 30% sucrose in 0.02 M PBS, pH 7.4, then embedded in OCT compound and stored in liquid nitrogen. 10-micrometer cryostat tissue sections were cut, collected on pre-cleaned glass slides, and air-dried for 20 minutes. Sections were prepared as follows at room temperature:

1. Wash in 0.02 M PBS, pH 7.4 (5 min).
2. Immerse in 50 mM glycine in 0.02 M PBS, pH 7.4 (10 min).
3. Wash in 0.02 M PBS, pH 7.4 (5 min).
4. Immerse in 5% normal goat serum (NGS) (Vector Laboratories; Burlingame, CA) (20 min).
5. Incubate in the primary antibody (MEC 13.3, M-20, or C-19) at dilutions of 1:25, 1:50, or 1:100 in 0.02 M PBS (60 min).
6. Wash in 0.02 M PBS, pH 7.4 (3 X 5 min).
7. Incubate in Nanogold-Fab' goat anti-rat or rabbit anti-goat secondary antibody diluted 1:50 or 1:100 in 0.02 M PBS, pH 7.4, 60 min.
8. Wash in 0.02 M PBS, pH 7.4 (3 X 5 min).
9. Postfix in 1% glutaraldehyde in 0.02 M PBS, pH 7.4 (2 min).
10. Wash in distilled water (3 X 5 min).
11. Develop 6 min with HQ silver in the darkroom.
12. Wash in distilled water (2 X 2 min each.
13. Fix in 5% sodium thiosulfate (1 min).
14. Wash in distilled water (3 X 5 min).
15. Postfix in 1% osmium tetroxide in sym-collidine buffer, pH 7.4 (10 min).
16. Wash in 0.05 M sodium maleate, pH 5.2 (5 min).
17. Stain with 2% uranyl acetate in 0.05 M sodium maleate buffer, pH 6.0 (5 min).
18. Rinse in distilled water (5 min).
19. Dehydrate in graded ethanols and infiltrate with a propylene oxide-eponate (Ted Pella, Redding, CA).
20. Embed by inverting eponate-filled plastic capsules over the slide-attached tissue sections.
21. Polymerize at 60°C for 16 hr.
22. Separate eponate blocks from glass slides by brief immersion in liquid nitrogen, cut thin sections with diamond knife and collect on uncoated 200-mesh copper grids.

Antibody against MEC 13.3 reacted strongly with the luminal and abluminal plasma membranes of the endothelial cells lining microvessels in normal tissues and in angiogenic vessels induced by a tumor and vascular endothelial growth factor (VEGF-A). Lateral plasma membranes were significantly less labeled. Conversely, M-20 strongly labeled the cytoplasmic face of the lateral plasma membranes, although sparing specialized junctions, and only weakly labeled the luminal and abluminal plasma membranes. Both antibodies stained a significant minority of vesicles and vacuoles comprising the vesiculovacuolar organelle (VVO). Neither was reactive in CD31-null mice. These results indicate that CD31 is distributed over the entire endothelial cell surface, except at specialized junctions, and in VVOs, but is not equally accessible to different antibodies in all locations.

Reference:

Feng, D.; Nagy, J. A.; Pyne, K.; Dvorak, H. F., and Dvorak, A. M.: Ultrastructural Localization of Platelet Endothelial Cell Adhesion Molecule (PECAM-1, CD31) in Vascular Endothelium. J. Histochem. Cytochem., 52, 87-102 (2004).

RN46 cells were grown to 70 % confluency. Antisense ODNs were complexed with sonicated cationic lipid vesicles consisting of SAINT-2 and DOPE (1:1). In preliminary work, Lipofectamine 2000, purchased from Invitrogen, was also employed, and antisense ODN complexes were prepared essentially as instructed by the manufacturer. Where indicated, 0.5 mol % N-(lissamine rhodamine sulfonyl)-PE (NRh-PE, Avanti Polar Lipids Inc) was included in the lipid mixture to monitor the fate of the lipid complex by confocal fluorescence microscopy. Antisense / cationic lipid complexes were prepared as follows: 15 nmol SAINT-2/DOPE vesicles or 1-3 microliters Lipofectamine 2000, diluted in 500 microliters of DMEM, was gently mixed (45 min) with 0.1 nmol ODNs, also diluted in 500 microliters of DMEM. The complexes were allowed to equilibrate for 20 min at room temperature, after which time they were immediately added to the cell cultures. After 6 h, the complexes were removed by washing with PBS. The cells were fixed with 4 % paraformaldehyde in PBS for 15 min, and permeabilized with 0.1 % Triton X-100 for 30 min. Subsequently, the cells were incubated with FluoroNanogoldstreptavidin for 2 h, washed three times with PBS, and immediately examined by epifluorescence microscopy. After examination, the cells were washed with 0.2 M sodium citrate, and enhanced with Nanoprobes HQ Silver . The cells were rinsed with PBS and postfixed with 1 % glutardialdehyde for 10 min. Finally, the samples were embedded in Epon, serially sectioned (60-nm thin sections), and examined at 60 kV.

Gold labeling was seen throughout the nucleoplasm, with little if any labeling in the nucleolus. Dense gold labeling was apparent on the ODN bodies, especially at their periphery. At higher magnification, gold labeling of ODNs in the nucleoplasm could be seen associated along the fibers of the nuclear matrix.

5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells were all efficiently downregulated following ODN delivery via a cationic lipid delivery system. These results demonstrate that the gene sequence per se, rather than intracellular factors, primarily determines the antisense effect. In addition, the delivery method is a relevant factor: antisense ODNs bind extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than steric blockage of mRNA impedes protein expression. The specificity of the antisense approach is reflected by the effective functional downregulation of the 5-HT1A receptor.

Reference:

Shi, F.; Visser, W. H.; de Jong, N. M.; Liem, R. S.; Ronken, E., and Hoekstra, D.: Antisense oligonucleotides reach mRNA targets via the RNA matrix: downregulation of the 5-HT1A receptor. Exp. Cell Res., 291, 313-325 (2003).

A polyclonal antibody was raised in rabbits against the synthetic peptide corresponding to residues 13 - 37, near the putative N-terminus of the Kv1.4 gene product. For pre-embedding immunoperoxidase and gold labeling for EM, freefloating sections were incubated in 10% NGS diluted in TBS for 1 h, then incubated for 48 h in polyclonal antibody (code no. K8D) at a final protein concentration of 1 - 2 mg/ml diluted in TBS with 1% NGS. Immunoperoxidase labeling was conducted after several washes in TBS by incubation for 2 h in biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:50 in TBS / 1% NGS, followed by avidin-biotin-peroxidase complex (ABC kit, Vector Laboratories) diluted 1:100 for 2 h at room temperature. Peroxidase enzyme activity was revealed using 3,3' - diaminobenzidine tetrahydrochloride (DAB; 0.05% in TB, pH 7.4) as chromogen and 0.01% hydrogen peroxide as substrate. For immunogold labeling, sections were incubated in Nanogold® goat anti-rabbit IgG diluted 1:100 in TBS containing 1% NGS, washed several times in PBS, then postfixed in 1% glutaraldehyde in the same buffer for 10 min, washed in double distilled water, and enhanced using HQ Silver. Both peroxidase-reacted and the gold-silver labeled sections were then treated with 1% osmium tetroxide in 0.1 M phosphate), block-stained with uranyl acetate, dehydrated in graded series of ethanol and flat-embedded on glass slides in Durcupan (Fluka) resin. 70-90 nm thick sections from regions of interest were mounted on 200-mesh nickel grids and stained on drops of 1% aqueous uranyl acetate followed by Reynoldss lead citrate.

At the light microscopic level, the Kv1.4 subunit showed a unique distribution pattern, localized in specific neuronal populations of the rat brain. Regions expressing the highest levels of Kv1.4 protein included the cerebral cortex, the hippocampus, the posterolateral and posteromedial ventral thalamic nuclei, the dorsolateral and medial geniculate nuclei, the substantia nigra and the dorsal cochlear nucleus. The Kv1.4 subunit was mostly diffusely distributed; to a lesser extent, it stained cell bodies and proximal dendrites, and Kv1.4 immunoreactivity was also detected in nerve terminals and axonal terminal fields. At the electron microscopic level, Kv1.4 was located postsynaptically in dendritic spines and shafts at extrasynaptic sites, as well as presynaptically in axon and active zone of axon terminals, in the neocortex and hippocampus. Kv1.4 channels are shown to be widely distributed in the rat brain, and activation would have a variety of modulatory effects on neuronal excitability.

Reference:

Lujan, R.; de Cabo de la Vega, C.; Dominguez del Toro, E.; Ballesta, J. J.; Criado, M.; Juiz, J. M.; Immunohistochemical localization of the voltage-gated potassium channel subunit Kv1.4 in the central nervous system of the adult rat. J. Chem. Neuroanat., 26, 209-224 (2003).

For labelling with anti-clathrin and anti-MIP26 antibody, a three-step Nanogold pre-embedding procedure was used. Vibratome slices of formaldehyde-fixed rat lenses were fixed in 4% paraformaldehyde50 mM PBS (pH 73) for 50 min at room temperature (RT), washed in PBS overnight at 4°C, then cut into thick slices, and incubated in a blocking solution containing either 1% BSA and 03% Triton X-100 in PBS for 2 hr at room temperature. The tissue slices were incubated with primary antibody diluted in the blocking solution overnight at 4°C. After several washes in PBS, the lens slices were incubated in biotinylated goat anti-mouse IgG secondary antibody (1:100) in the blocking solution for 25 hr at RT, washed in PBS, followed by Nanogold-streptavidin (1:10) in 1% BSA, 005%TritonX-100 in PBS overnight at 4°C. The slices were washed in PBS, fixed with 25% glutaraldehyde in PBS for 30 minutes at RT, washed in PBS and water, enhanced with HQ Silver for 15 minutes at 18°C, rinsed in water and cacodylate buffer, pH 73, then fixed in 01% osmium tetroxide in 01 M cacodylate buffer, pH 73, for 30 min on ice and processed for TEM. Thin sections (80 nm) were stained with uranyl acetate and Reynolds lead citrate.

A three-step post-embedding procedure was used for labeling with anti-actin antibody: lenses were fixed immediately in 4% paraformaldehyde - 025% glutaraldehyde in 01 M cacodylate buffer (pH 73) for 1 hr at room temperature. Lenses were cut into 300 micrometer thick slices, dehydrated through a series of graded ethanol and embedded in LR White resin in gelatin capsules. Thin sections were cut and collected on nickel grids. The sections were blocked in a solution containing 5% dry milk and 2% BSA in PBS for 1 hr at RT, incubated on droplets of chicken anti F-actin polyclonal antibody(1:15 dilution), with 2% BSAPBS, pH 73 for 2 hr at room temperature. Grids were washed with PBS, incubated 1 hr on droplets of biotinylated goat anti-rabbit IgG (diluted 1:150) with 2% BSA-PBS, pH 73, washed with PBS, and finally incubated for 1 hr on droplets of streptavidin-10-nm gold diluted 1:25 with 2% BSAPBS, pH 73, and washed with PBS and distilled water. Grids were stained with uranyl acetate and Reynolds lead citrate.

References: