UCSF

BrdU staining

BrDU immunohistochemical staining

Solutions that have to be prepared before use

  1. 4% Paraformaldehyde/PBS: 1.5ml PBS + 500ul 16% paraformaldehyde
  2. 0.3% Triton X-100: 6ul Triton + 2ml PBS
  3. PBS/T20: 50ul Tween 20: (10% in stock) + 10ml PBS
  4. PBS/T20/2% normal Goat Serum: 20ul Goat Serum + 1ml PBS/T20

Procedure

  1. Wash the chamber with PBS X3.
  2. Add 500 ul of 1mM BrdU(1:100) with PBS to chamber. Incubate at 37 C for 1-2 hours.
  3. Wash with PBS X3.
  4. Wash chamber with 500ul 4% paraformaldehyde/PBS. Incubate at RT for 15 min.
  5. Wash with PBS X3
  6. Permeabilize with 0.3% Triton X-100 at RT for 15 min.
  7. Add 500ul HCL (2M). Incubate at 37 C for half an hour.
  8. Suck HCL off. Neutralize with 0.1M Borate Buffer (pH=8.5) X3.
  9. Wash with PBS/T20 X3
  10. Add 500ul PBS/T20/2% Serum. Incubate at 37 C for 30 min.
  11. Suck off the mixture. Add anti-BrdU (1/200). Incubate at 37 C for 30 min.
  12. Wash with PBS/T20 X3.
  13. Add second antibody (1/500) Alexa Flour 568. Incubate at 37 C for 30 min.
  14. Wash with PBS X5.
  15. Mount the slide.