Silver: High-throughput immunostaining

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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
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