Immunocytochemistry:  Acetone Fixative

Materials:

  1. Cold acetone fixative
  2. 20 x phosphate buffered saline
  3. Peroxidase block reagent
  4. Remaining reagent in Histostain SP Kit

Methods:

  1. Prepare the frozen sections, store at -20 C until usage
  2. Remove sections from freezer and air dry for 20 minutes
  3. Circle tissue sections with hydrophobic marker (PAP PEN)
  4. Fix sections in cold, dry acetone at -20 C for 7.5 minutes
  5. Air dry sections x 10 minutes
  6. Block: Add 10% normal goat serem to surface of sections, keep in humidified chamber at room temperature for 60 minutes
  7. Dilute the primary antibody to 10 mg/ml and apply to surface of sections.  Leave over night at 4 C.
  8. Wash in 1 x PBS for 2 x 5 minutes
  9. Peroxidase block - soak in methanol/peroxidase reagent at room temperature for 30 minutes.
  10. Wash in 1 x PBS for 3 x 5 minutes.
  11. Apply secondary antibody at a dilution of 20 mg/ml for 30 minutes at room temperature.
  12. Wash in 1 x PBS for 3 x 5 minutes
  13. Add enzyme conjugate for 10 minutes at room temperature
  14. Wash 1 x PBS for 3 x 5 minutes
  15. Add 1 drop of the AEC substrate to 1 ml of water and apply to the surface of the section for 5 - 8 minutes, monitoring for color development
  16. Wash in distilled water x 2
  17. Counterstain with hematoxylin for 10 seconds
  18. Immediately wash in tap water x 2
  19. Mount the slides