TUNEL assay

PROTOCOL:•Deparaffinize and rehydrate slides:3 x 3´ Xylene3 x 2´ 100% ethanol1 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 Component32 μl TE10 μl 5X Terminal Transferase buffer3 μl 25 mM CoCl2 (comes with TdT)45 μl•Nucleotide Mix:1x ____x Component0.25 μl 1 mM Alexa 488 dUTP0.5 μl 1 mM dATP4.25 μl TE5 μl•Reaction Mix*:1x ____x Component45 μl Equilibration Buffer5 μl Nucleotide Mix1 μl TdT Enzyme [or TE]51 μl*Make up one half with enzyme and the other without, replacing TdT with TE.