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 -20^oC
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 4^oC
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