In Situ End Labelling (tunneling): 4/98

1. Bake 4-6um thick sections onto slides O/N at 55’C.

2. Deparaffinize sequentially in Hemo De Xylenes (3:1) 2X for 5 minutes.

3. Rehydrate in ETOH (100%, 100%, 95%, 70%, H2O) for 5 minutes each. Treat + control here*.

4. Proteinase K treatment: equilibrate at 37’C for 5 minutes in 10mM Tris (pH 7.5 @37’C) in copplin jar.

5. Treat for 15 minutes with 10ug/ml PK in above buffer. (for adult, non-fragile tissue only).

6. Wash in TBS 3X/RT/5 minutes. (1/2000)

Steps 7-9 (Optional):

7. Antigen Retrieval: In plastic cup, add 0.1M Citric Acid, pH 6.0 (made fresh daily).

Pack slides tightly to avoid bubbles (optional).

8. Microwave: power level ten, ~ 1 minute, until boils; power level one, 25 minutes; RT 5 minutes. Allow to cool to RT for 10 minutes before wash.

9. Wash with RT dH2O 2X/RT/5 minutes.

10. Equilibrate in TdT buffer 10 minutes at 37’C then proceed to label as below:

11. Incubate in TdT buffer + Enzyme emperically or for 20 minutes for the first time at 37’C.

12. Wash in excess with TBS.

13. Block in 10% non-fat dry milk in TBS/37’C/1 hour then cool to RT.

14. Dilute Ab 1:500 in Tris Triton buffer. (sheep anti-Dig alkaline Phosphatase, BMB).

15. Direct detect with Ab emperically or for 15-30 minutes at RT.

16. Wash 3X/RT/TBS.

17. Make Detection solution immediately before use (1:50 dilution in 0.1M Tris, pH 8.1).

18. Equlibrate in 0.1M Tris HCl, pH 8.1 for Vector Red or blue (pH 9.5 for NBT/BCIP).

19. Detect with Vector Red detection solution emperically but check after 5 minutes.

20. Wash off with dH2O to stop reaction when staining is complete.

21. If desired, counterstain with methyl green then destain in acetone + 0.5% acetic acid.

+ control is treated with 1/1000 dilution of promega Dnase RQ-1 solution/10 minutes/RT.