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HOME > Protocols > Histology > Staining protocols > Protocol for Chloramphenicol Acetyltransferase (CAT) staining of tissue

Protocol for Chloramphenicol Acetyltransferase (CAT) Staining of Tissue

  1. Cut tissues on cryostat
  2. Collect tissue sections on clean poly-l-lysine coated glass slides
  3. Dry for 24 hours at room temp (can be shorter)
  4. Fix in 100ml of 100% acetone at -20oC
  5. Wash three times with PBST
  6. Incubate slides for 20 minutes in 2% normal goat serum (NGS) in PBS
  7. Wash six times with PBST
  8. Add primary antibody: Digoxigenin anti-CAT in 2%NGS/1%BSA in PBS (pH 7.2). This should be titrated based on tissue expression.
  9. Add negative control 2% NGS in 1% BSA in PBS (pH 7.2)
  10. Incubate overnight at 4oC
  11. Wash six times with PBST
  12. Add 250-300ul of anti-Digoxigenin-POD fragment in 100mM tris/150mM NaCl and 1% NGS (pH 7.5)
  13. Incubate 60 minutes at room temperature in the dark
  14. Wash six times with PBST
  15. Add 300-300ul of DAB substrate
  16. Incubate 10 minutes at room temperature in the dark
  17. Rinse once with distilled water
  18. Mount slide

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