TUNELassay

PROTOCOL:
•Deparaffinize and rehydrate slides:
3 x 3´ Xylene
3 x 2´ 100% ethanol
1 x 2´ 95%, 80%, 70% ethanol (each)
1 x 5´ 1x PBS
•Microwave antigen retrieval:
4 x 5´ in microwave (600 ml of 10mM NaCitrate, pH 6)
Cool 20´. Wash 3 x 5´ in water. Wash 1 x 5´ in 1x PBS (Phosphate-Buffered Saline).
•Shake off/wipe off excess PBS and circle all sections with ImmunoEdge or PAP pen.
•Block 10 minutes in Equilibration Buffer at room temperature (50 μl/section).
•Add 50 μl of Reaction Buffer to each section and incubate at 37°C for 60-90 minutes.
[One section WITH TdT enzyme, one section without enzyme as a negative control]
•Soak slides in 1xSSC for 15 minutes at room temp to stop reaction.
•Wash 5 x 5´ in 1x PBS.
•Mount the sections in 3:1 Vectashield:DAPI. Coverslip and seal with clear nailpolish.
BUFFERS:
•Equilibration Buffer:
1x ____x Component
32 μl TE
10 μl 5X Terminal Transferase buffer
3 μl 25 mM CoCl2 (comes with TdT)
45 μl
•Nucleotide Mix:
1x ____x Component
0.25 μl 1 mM Alexa 488 dUTP
0.5 μl 1 mM dATP
4.25 μl TE
5 μl
•Reaction Mix*:
1x ____x Component
45 μl Equilibration Buffer
5 μl Nucleotide Mix
1 μl TdT Enzyme [or TE]
51 μl
*Make up one half with enzyme and the other without, replacing TdT with TE.