Colloidal gold conjugates

Ready-to-use blocking solutions containing normal inactivated sera or BSA for use with colloidal gold conjugates are available from Aurion (http //www. aurion.nl/). When labeling with protein A and protein G conjugates, this step can be omitted. 

Colloidal gold A suspension (or colloid) of submicrometer-sized particles of gold in a fluid, usually water. A colloidal gold conjugate consists of gold particles coated with a selected protein or macromolecule, such as an antibody, protein A or protein G. Because of their high electron density, the gold particles are visible in the electron microscope without further treatment. 

Incubate slides for 30 min at room temperature in colloidal gold conjugate diluted in TBS plus 1% BSA. 

Colloidal gold conjugated with streptavidin (see Note 3). 

Add ligand conjugated to the colloidal gold, i.e., colloidal gold conjugated with antibody to a specific receptor. 

Colloidal gold-antibody conjugates are also widely used, especially in indirect immunocytochemical methods. However, the stability of these gold conjugates is less than that for protein A, and depending on the antibody, the affinity may be less. Colloidal gold conjugated to avidin (see... 

Incubate grids for 30 min at room temperature in colloidal gold conjugated with protein A, protein G, immunoglobulin, or if a biotinylated secondary antibody was used, streptavidin (see Note 6). 

Excess NGS is removed, and the sections are covered with 100 pi of colloidal gold conjugated secondary antibody. The slide is placed on the polystyrene platform in the microwave oven and heated at 50% for 5 min the oven contains a water load of 200 ml of tap water. The sections are washed in three changes of 5 min each in Tris buffer n, rinsed with distilled water, and washed three times of 3 min each in distilled water. 

Fig. 1. Transmission electron micrograph of RBL-2H3 cells with colloidal gold conjugated to a monoclonal antibody against the IgE receptor. (A) Gold conjugate is localized primarily in coated pits. (B) Five minutes after exposing cells to antibody-coated gold, gold particles are localized in early endosomes. Bar = 0.5 pm.

The colloidal gold conjugate should be directed against the primary antibody and diluted to 1-5 pg/mL in PBS without calcium and magnesium plus 1%BSA. 

Ultrastnictural ISH has been applied to study different aspects of mRNA transport and localization and, to a lesser degree, of DNA, using primarily biotinylated or haptenated probes. Colloid-gold conjugated antibody or streptavidin is convenient as the detector system (Binder et al., 1986). 

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