Silver: High-throughput immunostaining

Detection of 14-3-3 in U2OS and HeLa cells by immunofluorescence

 * 1) Seed ~3000 cells in 384-well plates.
 * 2) Fix cells with 3% paraformaldehyde for 30 minutes at RT.
 * 3) Wash 1 time with PBS.
 * 4) Permeabilize cells with 0.5% Triton X-100 for 5 minutes.
 * 5) Wash 2 times with PBS.
 * 6) Block with 3% BSA in PBS for 30 minutes at RT (can leave O/N at 4C).
 * 7) Incubate with primary antibodies for 60 minutes.
 * 8) *Antibody solution should contain 1% BSA.
 * 9) *14-3-3σ monoclonal is diluted 1:5.
 * 10) Wash 2 times with 0.05% NP-40.
 * 11) Wash 2 times in PBS.
 * 12) Incubate with secondary antibodies and DAPI (all at 1:1000) for 1 hour.
 * 13) Wash 2 times with PBS.
 * 14) *Leave covered in PBS for storage at 4C and visualizing.

Reagents used:

 * 3% paraformaldehyde (100 mls PBS)
 * 3 g paraformaldehyde
 * 2 g sucrose


 * 0.5% Triton X-100
 * 20 mM Tris pH 7.4
 * 50 mM NaCl
 * 300 mM sucrose
 * 3 mM MgCl2


 * BSA 10mg/ml in PBS


 * 0.05% NP-40 in PBS