TUNEL assay

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.