Postembedding Immunolabeling

Postembedding Immunolabeling

1. Cut sections (>100 nm thick) on formvar coated Ni grids

2. Labeling on drops
a. 1x5 min. with 0.1MPBS (pH7.4) containing
i. 1%BSA
ii. 0.05% Triton x-100
iii. 0.05% Tween 20

b. 2 hr. @ RT and @4C overnight with Antibody in 0.01M PBS (pH7.4) containing
i. 1%BSA
ii. 0.05% Tween 20

c. Wash 3x1 min. on drops of 0.01M PBS

d. 1 Hr. @RT Incubation with secondary antibody conjugated with Au labels in 0.1M PBS containing
i. 1% BSA
ii. 0.05% Tween 20

e. Wash 3x1 min. on drops of 0.01M PBS

f. Allow grids to dry

3. Counter stain
a. 3% UA 4 min.
b. Lead citrate <1min.