Protocols: Post-Embedding Immunogold Method
L.R. white sections are collected on gold Veco grids.
If blocking is necessary for nonspecific labeling, buffer is supplemented with normal goat serum (2 - 10%) and bovine serum albumin (0.2 - 1%). We usually start with a 2% NGS and 0.2% BSA block and increase if necessary up to 10% and 1% respectively. Grids are floated face down on drops of blocking solution in tris buffered saline with 1.0% tween-20 and incubated for 60 min.
Grids are then transferred to drops of appropriately diluted primary antibody in tris-buffered saline, pH 7.6, with 1.0% tween-20. Grids are incubated in the refrigerator, approx. 4oC, overnight in a humidified chamber.
The following morning, grids and solutions are allowed to come to room temperature.
Grids are washed 10 X 1 min. each (or longer and more frequently, if needed) in buffer.
Transfer grids to appropriate gold-labeled secondary antibody (1:20) diluted in tris- buffered saline with 1.0% tween-20. First spin diluted gold conjugated antibody in a microfuge at middle speed for 30 seconds - 1 minute to get rid of gold clumps. Incubate for 60 mins. at room temperature.
Wash grids 10 X 1 min. each in buffer.
Wash grids 10 X 1 min. each in double-distilled H2O.
Counter stain (if desired) for E.M. with UA/Pb.
Tris-buffered saline, pH 7.6
0.15M sodium chloride
pH to 7.6
From: Spector et al., 1991. EMBO J. 10, 3467-3481.