Updated: February 12, 2002

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

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.

This procedure has been described by Tanner and co-workers, and is reported to give significantly higher densities of silver-enhanced gold particles than other methods. An example of the results is shown below:

Nanogold-labeled K+ Channel (77k)

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

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

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.
Wash with 0. 1 M sodium phosphate buffer, pH 7.4, 3 x 5 min each.
Blocking and permeabilize the cells with PBS with 5 % goat serum, 0.1% sodium azide and 0.1% Saponin for 1 hour.
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.
Wash with PBS with 1% goat serum and 0.1% sodium azide for 3-4 x 5 min.
Incubate with Nanogold -labeled Fab' anti-rabbit or mouse (depending on the primary antibody) secondary antibody conjugate ( 4 µl) in 1 ml of PBS with 1% goat serum and 0.1% sodium azide for 1 hr, room temp.
Wash with PBS containing 1% goat serum with 0.1% sodium azide, once, then PBS, twice.
Fix with 2% glutaraldehyde in PBS for 30 minutes.
Wash 3 times in PBS. Store overnight.
Next day:
Wash with water thoroughly.
Perform silver enhancement (HQ Silver enhancement kit, Nanoprobes, NY).
Wash in water. Check under LM carefully; only process the promising specimens for EM.
Wash in 0.1M phosphate buffer, pH 7.4.
0.2% OsO₄ in 0,1 M phosphate buffer for 30 minutes.
Wash, stain with uranyl acetate, dehydrate in ethanol, and embed.

References

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).
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

In the Proceedings of Microscopy & Microanalysis 2001, Grondin and Beaudoin report a new pre-embedding immunogold method which gives a very high signal combined with very good ultrastructural preservation. It was demonstrated by labeling NADPase type 1 (a membrane protein) in confluent endothelial cells:

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.
Permeabilize with 50 % methanol at -20°C for 5 minutes.
Wash with phosphate-buffered saline containing 0.1 % Tween-20 (PBST).
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).
Incubate at 4°C overnight with primary antibody diluted 1 : 300 in PBSB.
Wash three times in PBST.
Incubate 1 hour at room temperature with Nanogold-labeled secondary antibody diluted 1 : 300 in PBSB.
Wash three times in PBST.
Fix 1 hour in 1.2 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, with 5 % sucrose.
Wash with water.
Enhance using GoldEnhance LM (6 minutes).
Post-fixation in 2 % osmium tetroxide and 2 % potassium ferricyanide in the same buffer.
Dehydrate, embed in Epon 812, section and stain with lead citrate before examination in the electron microscope.

Reference:

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.

For more information contact:

Email us at nano@nanoprobes.com.

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