High-Density, Reliable Pre-Embedding Nanogold® Labeling Procedures

Contents

• Procedure using HQ Silver Enhancement
• Procedure using Gold Enhancement

Procedure using HQ Silver Enhancement

• Higher density gold labeling than with other methods.
• Very high specificity.
• Maximum resolution.

Nanogold®:-Fab' goat anti-rabbit IgG (Catalog # 2004) labeling the K⁺ channel Kv2.1 subunit in rat brain, followed by HQ Silver (Catalog # 2012) enhancement. Note high density and specificity of immunostaining, even elucidating subunit localization to cytoplasmic side of cell membrane and outer stacks of the Golgi; axons and terminals are clearly negative. Work done by J. Du, J.-H. Tao-Cheng, P. Zerfas, and C. J. McBain, NIH. See Neuroscience, 84, 37-48 (1998). Bar = 1 micron.

Materials and Reagents

Sodium phosphate buffer: 0.1 M sodium phosphate, pH adjusted to 7.4.
• Phosphate-buffered saline (PBS) buffer: 0.02 M sodium phosphate buffer with 0.15 M sodium chloride, pH adjusted to 7.4.
• Phosphate-buffered saline (PBS) buffer: 0.02 M sodium phosphate buffer with 0.15 M sodium chloride, pH adjusted to 7.4, containing (a) 5 % bovine serum albumin and 0.05 to 0.1 % sodium azide; and (b) 1% goat serum and 0.1% NaN3 for 3-4 X 5 min
• Glutaraldehyde and paraformaldehyde.
• HQ Silver reagent (Nanoprobes).
• Deionized or distilled water.

Procedure

1. Fix for ~45 minutes (for monolayer cultures) with one of the following: (1) 4% paraformaldehyde in 0.1M sodium phosphate buffer, pH 7.4, or (2) 2% paraformaldehyde with 0.05% - 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4.

2. Wash with 0. 1 M sodium phosphate buffer, pH 7.4, 3 x 5 min each.

3. Blocking and permeabilize the cells with PBS with 5 % goat serum, 0.1% sodium azide and 0.1% Saponin for 1 hour.

4. Incubate with primary antibody made in PBS with 5% normal goat serum, 0.1% saponin and 0.1% sodium azide for 1 hour at room temp.

5. Wash with PBS with 1% goat serum and 0.1% sodium azide for 3-4 x 5 min.

6. Incubate with Nanogold -labeled Fab' anti-rabbit or mouse (depending on the primary antibody) secondary antibody conjugate ( 4 l) in 1 ml of PBS with 1% goat serum and 0.1% sodium azide for 1 hr, room temp.

7. Wash with PBS containing 1% goat serum with 0.1% sodium azide, once, then PBS, twice.

8. Fix with 2% glutaraldehyde in PBS for 30 minutes.

9. Wash 3 times in PBS. Store overnight.

   Next day:

10. Wash with water thoroughly.

11. Perform silver enhancement (HQ Silver enhancement kit, Nanoprobes, NY).

12. Wash in water. Check under LM carefully; only process the promising specimens for EM.

13. Wash in 0.1M phosphate buffer, pH 7.4.

14. 0.2% OsO₄ in 0,1 M phosphate buffer for 30 minutes.

15. Wash, stain with uranyl acetate, dehydrate in ethanol, and embed.

References

1. Tanner, V. A., Ploug, T., and Tao-Cheng, J.-H. Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM Immunocytochemistry for cell cultures. J. Histochem. Cytochem., 44, 1481-1488 (1996).
   • Abstract (from JHC Online).

2. Du, J.; Tao-Cheng, J.-H.; Zerfas, P., and McBain, C. J. The K+ channel, Kv2.1, is apposed to astrocytic processes and is associated with inhibitory postsynaptic membranes in hippocampal and cortical principal neurons and inhibitory interneurons. Neuroscience, 84, 37-48 (1998).
   • Abstract (from Medline).

Procedure using Gold Enhancement

1. Fix cells in situ for 3 hours with a freshly prepared and filtered solution of 1 % l-lysine, 4 % paraformaldehyde, 0.04 % glutaraldehyde and 0.25 % sodium metaperiodate in 0.04 M sodium cacodylate buffer, pH 7.4.

2. Permeabilize with 50 % methanol at -20°C for 5 minutes.

3. Wash with phosphate-buffered saline containing 0.1 % Tween-20 (PBST).

4. Block unspecific labeling with phosphate-buffered saline containing 0.1 % Tween-20, 2 % goat serum, 1 % bovine serum albumin, 0.45 % fish gelatin, and 0.4 % glycine (PBSB).

5. Incubate at 4°C overnight with primary antibody diluted 1 : 300 in PBSB.

6. Wash three times in PBST.

7. Incubate 1 hour at room temperature with Nanogold-labeled secondary antibody diluted 1 : 300 in PBSB.

8. Wash three times in PBST.

9. Fix 1 hour in 1.2 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, with 5 % sucrose.

10. Wash with water.

11. Enhance using GoldEnhance LM (6 minutes).

12. Post-fixation in 2 % osmium tetroxide and 2 % potassium ferricyanide in the same buffer.

13. Dehydrate, embed in Epon 812, section and stain with lead citrate before examination in the electron microscope.

Grondin, G., and Beaudoin, A. R.: A New Pre-Embedding Immunogold Method that Permits to Obtain a Very High Signal with a Very Good Ultrastructure. Microsc. Microanal., 7, (Suppl. 2: Proceedings) (Proceedings of the Fifty-Ninth Annual Meeting, Microscopy Society of America); Bailey, G. W.; Price, R. L.; Voelkl, E., and Musselman, I. H., Eds.; Springer-Verlag, New York, NY, 2001, pp. 1044-1045.

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Catalog: Nanogold®-Antibody conjugates
Product Information: Nanogold®-Antibody conjugates