apoptosis detection method

SHO1 sho1 at aol.com
Thu Mar 30 18:40:06 EST 1995


After several requests, I have decided to post this cookbook method.

If you have any questions, please call Jing Xu 516-663-2034.

TUNEL


1. Induce Apoptosis by your favorite treatment.
 
2. Rinse the cells with ice-cold PBS

3. Fix the cells in 4% formaldehyde or 10% buffered formalin for 10
minutes.

4. Permeabilize the cells in 0.5% Triton X100 for 5 minutes, wash in H2O x
4.

5. Treat with micrococcal nuclease for positive control (10 units/ml or
100 units/ml) for 30 minutes at room temperature,  then wash in water for
4 times

6.  TdT buffer equilibration with 1X TdT buffer
5-10 minutes at room temperature (up to 30 minutes) 

7. TdT enzyme reaction:  ea x ________ slides at 37xC for 1 hour.

    ea (40ul)   _______ x ea
________________________________________________________
5x TdT buffer  8 ul
5x CoCl2          8 ul
DIG-dUTP   2 ul (2 nmol)
dATP              0.2 ul (20 nmol)
TdT  Enzyme  2 ul (50 U)
H2O
_________________________________________________________
Total    40 ul

8. Immerse in 10mM EDTA for 10 minutes.

9. Wash with PBS x 2, 10 minutes each

10. Wash in TBS for 10 minutes

11. Blocking in TBS containing 1% BSA,  30 minutes at  room temp.

12. Incubate with 1: 500 anti-DIG-alkaline phosphatase antibody in 1%
BSA/TBS  for 1 hr at 37xC

13. Wash sample in TBS x 3 times, 5 minutes each

14.  Wash sample in Alkaline Phosphatase buffer for 15 minutes.

15. Develop with 45 ul NBT,  35 ul BCIP each 10 ml AP buffer.
     0.5 hr in the dark at room temp.

16. Wash  with water and mount  

 
************************************************************************  

The following reagents are purchased from Boehringer Mannheim Corporation:

Terminal transferase: cat. no. 22o 582 (contains 5x reaction buffer and
cobalt chloride)

dATP: cat. no. 1051 440

DIG-dUTP: cat. no. 1093 088

anti-DIG-alkaline phosphatase: cat. no. 1093 274                          
                                       



Best of luck!!

-Stuart



Stuart Horowitz
CardioPulmonary Research Institute
Email: sho1 at aol.com
Ph: 516-663-3917 Fax: 516-663-8874



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