View Cart   647 FluoroNanogold™ for Super-Res! Join our Mailing List

Nanoprobes Protocol:

High-Density, Reliable Pre-Embedding
Nanogold® Labeling Protocol
Using Gold Enhancement
with GoldEnhance™ EM Plus

Related products:


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.

Protocol:

  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 two times with water.
  11. Enhance using GoldEnhance EM Plus (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.
  14.  

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.

 


See also

 


 

Need help? Drop us a line:
Logo (14k) Nanoprobes, Incorporated
95 Horseblock Road, Unit 1, Yaphank, NY 11980-9710
Tel: (877) 447-6266 (Toll-Free in US) or (631) 205-9490  |  Fax: (631) 205-9493
[email protected] | www.nanoprobes.com

Other Applications

Product Applications

Research Applications

 

View Cart     Nanoprobes.com
© 1990-2017 Nanoprobes, Inc. All rights reserved. Sitemap