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.
   • Antibody solution should contain 1% BSA.
   • 14-3-3σ monoclonal is diluted 1:5.
8. Wash 2 times with 0.05% NP-40.
9. Wash 2 times in PBS.
10. Incubate with secondary antibodies and DAPI (all at 1:1000) for 1 hour.
11. Wash 2 times with PBS.
    • 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