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                                            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.

                                             


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