WangLab:Immunostaining General

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Protocol for Immunostaining

  1. 3X wash of the cells with ice-cold PBS.
  2. Fix the cells with 4% paraformaldehyde at RT for 15min.
  3. 3x 5min/each wash with PBS in RT.
  4. Permeabilize with 0.1% TX-100 in PBS for 15-30 min, at RT
  5. Rinse thoroughly with PBS, 3x wash 5min
  6. Incubate with 3% goat normal serum or 1% BSA for 20 min, at RT
  7. Wash 3x PBS, 5 min/each, at RT
  8. Incubate primary antibody (1:100, 10 ug/ml) in PBS at RT for 2 hr or 4oC O/N,.
  9. Wash 3X PBS, 5min/each, at RT
  10. Incubate secondary antibody conjugated with fluorescence dye (1:200) in PBS, at RT for 1 hr
  11. 3x PBS wash, 5 min/each, at RT
  12. Mount with anti-photobleaching reagent, seal the slide/dish
  13. Imaging