'A very reliable pre-embedding immunogold method'
Dr. Susan Cheng and co-workers at NINDS have refined their pre-embedding procedure for the most uniform, consistent and reliable pre-embedding labeling, using Nanogold®-Fab' and HQ Silver.

What our users have found with this protocol:

• 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 nanoparticles than other methods. An example of the results is shown below:

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.

Protocol

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).
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).

See also

• Pre-embedding Nanogold® Labeling Protocol
  with Enhancement by GoldEnhance™ EM Plus