UCSF

IF paraffin slides (double staining)

IF Staining with Paraffin Section Slide (Double Staining)

Procedure

  1. Heat slides in an oven at 60°C for 45 minutes. (usually do a day before)
  2. Deparaffinize/hydrate using the following series of washes:
    1. Xylenes 3X 5 min
    2. 100% EtOH 2X 2 min
    3. 95% EtOH 2X 2 min
    4. 80% EtOH 1X 2 min
    5. 70% EtOH 1X 2 min
    6. dH2O 2X 3 min + shaker
    7. PBS 1X 5 min + shaker
  3. Microwave slide in boiling 10mM Sodium Citrate Buffer. Fill chamber to brim with buffer. Microwave 10 minutes on high power in buffer and allow 20 minutes to cool down at RT.
  4. Fix with 200ul of 4% paraformaldehyde/PBS for 20 min at RT (fix time)
  5. Wash with PBS
  6. Permeabilize with 0.1% Triton x-100/PBS for 3 min at RT (fix time)
  7. Wash with PBS
  8. Treated with goat serum/PBS (1:200) for 30 min at RT.
  9. Wash with PBS
  10. Added primary Antibody in goat serum/PBS for 1 hr at RT
  11. Wash with PBS
  12. Added goat anti-rabbit 594 or anti-mouse for 30 min at RT.
  13. Wash with PBS
  14. Treated with goat serum/PBS (1:200) for 30 min at RT.
  15. Wash with PBS
  16. Added 2nd primary Antibody in goat serum/PBS for 1 hr at RT
  17. Wash with PBS
  18. Added 2nd goat anti-rabbit 594 or anti-mouse for 30 min at RT.
  19. Added goat anti-rabbit 594 or anti-mouse for 30 min at RT.
  20. Mounting the slide with reagent contain DAPI.
  21. Store at dark box at 4ºC. Good for one week.