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"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."Janet Duerr, Ohio University
ImmunoGold reagents offer excellent tissue penetration due to their small particle size (Dixon et al., 2015). ImmunoGold colloidal gold reagents are available for transmission (TEM) and scanning electron microscopy (SEM) (EM Grade 6, 12 and 18 nm), or for brightfield microscopy or immunoblotting (LM Grade 4 nm).
The EM Grade is distinguished from other commercial preparations by careful separation of monomeric particles from small aggregates using ultracentrifugation in density gradients. The resulting monomeric colloidal gold-protein complexes are recommended for multiple labeling applications, as different antigenic sites can be distinguished by particle size. The complexes are suspended in sterile-filtered buffer containing stabilizers and a preservative.
The 4 nm size may be used for electron microscopy in studies that require smaller particles since they are relatively uniform in size (coefficient of variation less than or equal to 15%), though small aggregates are not removed from this grade. The 4 nm particles are not suitable for multiple labeling with EM Grade reagents, since size uniformity is paramount and aggregated material may be mistaken for a larger particle.
Silver enhancement allows the excellent penetration properties of ImmunoGold reagents to be used with light microscopy. The gold particles act as a nucleation site for the silver ions, which accumulate around the particle until enough contrast is generated to be visualized (Dixon et al., 2015). A detailed protocol for silver enhancement, using reagents that are easily prepared in the laboratory, is provided with all orders for LM Grade products. Alternatively, silver enhancement kits are commercially available.
Signal intensity is relatively independent of particle size when silver enhancement is used, so all particle sizes may be used for light microscopy or immunoblotting. For light microscopy, 4 nm particles (LM Grade) may penetrate tissues better than larger particles.
All LM Grade colloidal gold-protein complexes are freeze-dried in buffer with stabilizers and a preservative. After reconstitution, they may be frozen in aliquots for extended storage.
Figure 2. Immunolabeling for collagen type IV in normal human skin using ImmunoGold reagents 4 nm with silver enhancement. Prof. Jurgen Roth, Dept. Path. University of Zurich.
Dixon, A. R., Bathany, C., Tsuei, M., White, J., Barald, K. F., & Takayama, S. (2015). Recent developments in multiplexing techniques for immunohistochemistry. Expert Review of Molecular Diagnostics, 15 (9), 1171–1186. http://doi.org/10.1586/14737159.2015.1069182.
Timothy D. Blalock, et al; Functions of MUC16 in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(10):4509-4518