Immunostaining for cultured cell

Immunostaining for cultured cell

Material

Fixation

  1. Use 1 ml sol/dish in following procedure.
  2. Wash by PBS twice.
  3. Fix by 2% PFA at RT for 5 min.
  4. Wash by 0.01% Triton twice.
  5. OPTIONAL: Save dish with 0.01% Triton at 4C (cold room).
  6. Permeabilize by 0.2% Triton at RT for 5 min.
  7. Wash by 0.01% Triton once.
  8. Mask circumference of dish by PAP pen.
  9. Use 150 ul sol/dish in following procedure.
  10. Blocking by 1% BSA at RT for 30 min.

Primary antibody

  1. Prepare primary antibody sol in 1% BSA at appropriate concentration.
    See Antibody.
  2. Suck 1% BSA.
  3. Add primary antibody sol.
  4. Incubate at RT for 1 h or at 4C for o/n.

Secondary antibody

  1. Prepare secondary antibody sol in 1% BSA at appropriate concentration.
    See Antibody.
  2. Wash by 0.01% Triton twice.
  3. Add secondary antibody sol.
  4. Incubate at RT for 1 h.
  5. Wash by 0.01% Triton twice.
  6. Add 1/10,000 DAPI.
  7. Incubate at RT for 5 min.
  8. Suck DAPI.
  9. Add 1-2 drop of mounting medium.
  10. Enclose by cover glass.