Cells grown on glass coverslips are immunolabelled prior flat-embedding. For immunolabelling, cells are fixed with PLP-fixative and permeabilized by saponin. For detection of immunolabelling, we use Fab-fragments of secondary antibodies conjugated with 1.4 nm gold particles (Nanoprobes). The use of small gold particles and Fab-fragments allows efficient diffusion in fixed cells. Gold particles are then enhanced with HQ-silver enhancement kit (Nanoprobes) and stabilized by gold toning prior to flat embedding.

Silver enhanced immunogold labelling of nsP1 protein in Semliki forest virus -infected HeLa-cells on the cytoplasmic side of the limiting membrane of virus-induced cytoplasmic vacuoles (CPVs). Arrows indicate spherules, which are the sites of synthesis of viral RNA. (Salonen et al., 2003, J Virol 77:1691-1702). Bars 2 µm (upper figure) and 500 nm (lower figure).

Silver enhanced immunogold labelling

Immunogold labelling of Galactosyl transferase enzyme localizing to trans-Golgi cisternae (left) or Golgi matrix protein GM130 localizing to the cytoplasmic side of cis and medial Golgi cisternae (right) in NRK cells (E. Jokitalo).

The effect of enhancement time becomes clear when comparing these three Golgi protein labellings (E. Jokitalo).

Gold toning stabilizes the silver precipitate. Without gold toning, only some of the precipitate formed during long enhancement time persists on the section. Immunolabelling is against Golgi matrix protein GM130 on NRK-52E cells (O.Välimäki).