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.