Immunolabeling

We employ two different methods for imunolabelling. The first one involves Cryo embedding, sectioning and labeling, while the second one is pre-embedding labelling where cells are grown on cover slips and the imunolabelling is performed on the whole cells before standard epon embedding.

Immunolabeling of cryo-sections (Tokuyasu method)

Cells are

• fixed with formaldehyde/glutaraldehyde mixture
• embedded in gelatine
• infiltrated with sucrose for cryoprotection
• frozen by immersion in liquid nitrogen

Ultrathin sections are cut with cryo-ultramicrotome at -120 °C and picked on carbon/pioloform-coated grids. Sections on grids are immunolabelled using secondary antibodies conjugated with 5, 10 or 15 nm gold particles (British BioCell) or protein A-conjugated with 5, 10 or 15 nm gold particles (University of Utrecht, School of Medicine, Departure of Cell Biology).

Pre-embedding immunolabelling

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 (NanoprobesOpens in a new tab). 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.