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1 Technical Bulletin DeadEndTM Fluorometric TUNEL System G3250 / 2015 3 TEL:03-3669-7980, 1 DeadEndTM Fluorometric TUNEL System G3250 I.. 1 II.. 4 III.. 4 A..4 B.. 4 IV.. 4 A..4 B..6 C..7 D..8 E. DNas e ..8 Anisomycin HL-60 ..9 VI.. 12 VII.. 13 VIII.. 14 IX.. 16 I. DeadEndTM Fluorometric TUNEL System G 3250 in situ non- RI DeadEndTM Fluorometric TUNEL System DNA blebbing 2001 11

2 AIDS T (1-8) TEL:03-3669-7980, 2 (9,10) DNA (11 14) DNA 180 200bp DNA 180 200pb DNA (15) DeadEndTM Fluorometric TUNEL System Terminal Deoxynucleotidyl Transferase(rTdT) DNA 3 -OH fluorescein-12-dUTP(a) DNA TUNEL TdT-mediated dUTP Nick-End Labeling rTdT fluorescein-12-dUTP DNA 1 2 3 (cytospin)

3 PBS 4 Triton X-100 Proteinase K Equilibration Buffer Plastic Coverslip Equilibration Buffer Nucleotide Mix rTdT Enzyme Plastic Coverslip 37 C 1 Plastic Coverslip 2 SSC propidium iodide propidium iodide DAPI( ) (propodium iodide) (DAPI) fluorescein-12-dUTP 1. DeadEndTM Fluorometric TUNEL System DNA fluorescein-12-dUTP TEL:03-3669-7980, 3II.

4 DeadEndTM Fluorometric TUNEL System 60 G 3250 Equil ibration Buffer 300 l Nucleotide Mix (6 50 l) 3x20 l TdT (Terminal deoxynucleotidyl Transferase) Enzyme (3 600u) 70ml 20X SSC 10mg Pr oteinase K 60 Plastic Coverslips Equilibration Buffer rTdT Enzyme Proteinase K 15 20 C Nucleotide Mix 15 20 C 20 SSC Proteinase K 1ml Proteinase K buffer VII Buffer Proteinase K solution 10mg/ml Proteinase K solution 20 C 6 III.

5 A. Nucleotide Mix Nucleotide Mix Nucleotide Mix B. Equilibration Buffer IV. DeadEndTM Fluorometric TUNEL System VII PBS propidium iodide Sigma P4170 SlowFade Light Anti-Fade Kit Molecular Probes S746 1 VECTASHIELD Vector Labs H- 1000 VECTASHIELD +DAPI Vector Labs H- 1200 1 - * (Polysciences 18814) PBS 4 - * (Polysciences 18814) PBS Note.

6 70 Tr iton X-100 PBS Tr iton X-100 PBS 5mg/ml BSA DNase I RQ1 RNase-Free DNase M6101 20mM EDTA pH DNase buffer DNase-free RNase A TEL:03-3669-7980, 4 4 - * (Polysciences 18814) PBS Note: 100 95 85 70 50 NaCl Proteinase K buffer DNase I DNase I buffer -L- Poly-PrepTM Slides Sigma P0425 Superfrost Plus Glass Slides Fisher 12- 550-15 Lab- Tek Chamber Slides Nunc 177380 DNas e I 37 C 37 C 37 C -L- Poly-PrepTM Slides Sigma P 0425 Superfrost Plus Glass Slides Fisher 12- 550-15

7 DNase I A. -L- -L- : 50 100 l w/v -L- Sigma P9155 Sigma P8920 10 -L- 30 60 -L- TEL:03-3669-7980, 5 : Lab-Tek PBS 2 DeadEndTM Fluorometric TUNEL System 1.

8 4 PBS 4 C 25 Note: - 2. PBS 5 Note: 2 20 C 70 4 C PBS 2 3. PBS Triton X-100 5 4. PBS 5 PBS Note: 4 DNA DNase I DNase I 5. 100 l Equl ibration Buffer 5 10 6. Nuc leotide Mix 1 rTdT rTdT 50 l 5cm2 Note:Nucleotide Mix rTdT 1 ( ) rTdT 50 l ( )

9 Equilibration Buffer Nucleotide Mix rTdT Enzyme 45 l 5 l 1 l _____ _____ _____ _____ l _____ l _____ l rTdT _____ l : rTdT Enzyme 45 l Equilibration Buffer 5 l Nucleotide Mix 1 l D. W. 1 50ul 7 16 : 7. 5cm2 50 l rTdT Note: TEL:03-3669-7980, 6 !

10 : 7 8. 37 C 60 9. 20 SSC 10 40ml 2 SSC 2 SSC 15 ! : 20 SSC 10. PBS 5 2 3 fluorescein-12-dUTP 11. PBS 1 g/ml 40ml propidium iodide 15 Propidium iodide DAPI+VECTASHIELD Vector Labs H- 1200 ) 16 12. 5 2 3 13. 14. 16 Anti-Fade solution Molecular Probes S74 61 1 15.