Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography

Abstract

Immunoelectron microscopy using ultrasmall gold markers is a very sensitive method to detect molecules at high resolution. In order to discriminate the gold particles in the electron microscope, enlargement of gold particles is necessary. So far, mostly silver ions were used for deposition onto the surface of gold grains. In our study, we tested the selective deposition of gold instead of silver ions to enlarge gold particles. This was performed following immunogold detection of DNA at the surface of ultrathin sections embedded in the acrylic resin LR White (postembedding approach). Morphometric analysis of the distribution of DNA in human spermatocytes revealed that the method offers very good specificity and sensitivity and therefore is a good alternative to the use of silver for signal enhancement. This technique was also applied to the detection of ribosomal genes in human testis at the electron microscopic level by in situ hybridization. Ribosomal genes were localized in peri-and intranucleolar chromatin as well as in the dense fibrillar component of nucleoli.

Focal adhesion interactions with topographical structures: a novel method for immuno‐SEM labelling of focal adhesions in S‐phase cells

“…A combination of a soluble silver compound and a reducing agent induces the deposition of silver onto the gold probe. This process, termed autometallography enhancement (Christensen et al , 1992; Weipoltshammer et al , 2000), permits the user to benefit from the low steric hindrance of small colloidal gold markers and the increased sample penetration, whilst still being able to visualize the markers at low magnification. The 1.4 nm gold colloid retains rapid nuclear penetration and high label efficiency (Richards et al , 2001) yet is available following antigen binding for subsequent enlargement via silver deposition.…”

Caveolin‐1 and Rac regulate endothelial capillary‐like tubular formation and fenestral contraction in sinusoidal endothelial cells

“…Pre‐embedding nanogold immunolabelling with silver enhancement was performed according to published protocols (18, 19). The LSECs were grown to near confluence and were given fresh medium containing collagen or Matrigel.…”

An improved method for nanogold in situ hybridization visualized with environmental scanning electron microscopy

“…First, metallic substrates and surfaces can obscure detection of nanogold-labelled cells imaged with backscattered electron microscopy (Richards et al, 2001). Second, metallic surfaces can interfere with the hybridization reaction by causing non-specific precipitation of nanogold (Humbel et al, 1995;Weipoltshammer et al, 2000). Because many geomicrobiological systems of interest have a high concentration of metal substrates (i.e.…”