Immunofluoresence

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Reagents

  • PHEM Buffer (25 mM HEPES, 10 mM EGTA, 60 mM PIPES and 2 mM MgCl, pH 6.9 – only if using paraformaldehyde)
  • Neutral buffered formalin
  • Cold PBS
  • 100 mM Glycine in PBS
  • 0.1% Triton X-100 in PBS
  • Blocking Solution: 1% BSA and 1% ovalbumin in PBS
  • Vectashield

Protocol

  1. Prepare Cells (For COS or the like, plate the day before to reach ~50%; for 3T3-L1 split one large plate into 12 wells (6 well plate, 2mL per well) at FBS day 1 and recover onto ethanol sterilized glass coverslips for 4 days in DMEM/PGS/FBS)
  2. Treat cells as required
  3. Wash cells twice with 2 mL cold PBS then fix for 10 min at RT
  4. Use neutral buffered formalin, 4% paraformaldehyde in PHEM or ice cold 10% methanol
  5. Wash twice with PBS
  6. Add 200 uL of 100mM Glycine in PBSfor 5 min to quench
  7. Wash once with PBS
  8. Permeabilize for 10 min with Triton X-100 (0.1% in PBS)
  9. Wash three times with PBS (5 min each)
  10. Block for 1-2h with 200 uL of BSA/Ovalbumin (1% of each in PBS) blocking solution
  11. Incubate overnight with primary antibody in blocking solution
  12. Wash coverslips 3 times 10 minutes with PBS (5 min each)
  13. Incubate in 500X secondary solution
  14. Wash coverslips 3 times 10 minutes with PBS (5 min each)
  15. Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish