Prepare clean labeling surface by laying down a long piece of parafilm on a wet bench and pressing it down to make it adhere tightly.
Quench unreacted aldehyde groups by placing grids on drops of 0.1M NH4Cl in PBS for 10 minutes @ RT.
Block non-specific binding sites by transferring grids on drops of 1% fish skin gelatin in PBS, for 20 minutes @ RT.
Prepare moist chamber using large Petri dish and pieces of filter paper soaked with water. Incubate grids on 10µl drops of primary antibody diluted in PBS/1% FSG for 30 min @ RT.
Wash 4 x in PBS (5 minutes total).
Label grids with secondary antibody (if necessary) for 30 minutes @ RT.
Wash 4 x in PBS (5 minutes total).
Label grids with Protein A Gold for 30 min @ RT.
Wash 4 x in PBS (5 minutes total).
Fix in 1% glutaraldehyde in PBS for 5 min.
Wash grids 5 x with distilled water.
Stain grids with 2% neutral uranyl acetate for 10 min in the dark.
Wash 3 x quickly on drops of distilled water.
Transfer grids to drops of 1.8% methylcellulose / 0.5% uranyl acetate on ice. After 2 quick rinses, transfer grids on final drops and incubate 10 min.
Pick up grids with special metal loops, blot excess methyl cellulose and air dry.
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