WangLab:Immunostaining Biotin

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Protocol for the Immunostaining of Biotin-Streptavidin System

  1. Fix the cells with 2-4% paraformaldehyde containing 1mM Na3VO4 at RT for 15-30min.
  2. 3x 5min/each wash in PBS containing 1mM Na3VO4.
  3. Permeabilize with 0.1-0.5% TX-100 in PBS containing 1mM Na3VO4 for 15-30 min, at RT
  4. Apply 1-2 drops of the streptavidin (molecular probes, E-21390), incubate for 15-30min at RT
  5. Rinse thoroughly with PBS containing 1mM Na3VO4, 1x wash 5min
  6. Apply 1-2 drops of biotin, incubate for 15-30min, at RT
  7. Rinse thoroughly with PBS containing 1mM Na3VO4, 1x wash 5min
  8. Incubate with 3% goat normal serum or 1% BSA for 20 min, at RT
  9. Wash 1x PBS, 5 min/each, at RT
  10. Incubate primary antibody (1:100, 10ug/ml) in (optional, 0.1%TX-100, 1% goat normal serum), at RT for 2 hr or 4oC O/N, in PBS containing 1mM Na3VO4.
  11. Wash 3X PBS, 5min/each, at RT
  12. Incubate biotinylated anti-mouse (from goat) (1:200 or 1:100) in (optional, 0.1%TX-100) PBS, at RT for 1 hr
  13. 3x PBS wash, 5 min/each, at RT
  14. Incubate FITC-streptavidin (1:200) in PBS, at RT for 30 min
  15. 3x PBS wash, 5min/each, at RT
  16. Optional: double staining, continue routine immunostaining if primary abs are from Rabbit.
    • Caution! If primary Abs are fromGoat, do Goat Staining first and keep 1mM Na3VO4 in all the solution used.
  17. Mount