Chapter 3
In Situ Nick End-labeling: light Microscopical
SHIGEYOSHI OBA AND TAKEHIKO KOJI
Introduction
It has been postulated that in apoptotic cell death, the first biochemical event is single-strand DNA breaks followed by doublestrand DNA cleavage in the linker regions between nucleosomes
(Peitsh 1993). TUNEL (Terminal transferase-mediated dUTPbiotin nick end labeling) is a convenient molecular histochemical tool to detect these single-DN A breaks. Briefly described, this
tool is based on repeated addition of biotin-dUTP at the 3-hydroxy ends of DNA of tissue by terminal deoxynucleotidyl transferase and detection of these additional biotin-dUTP by HRP labeled anti-biotin antibody. You can use digoxigenin-dUTP or
fluorescent conjugated dUTP instead of biotin-dUTP.
Outline
The entire protocol of TUNEL is outlined in Fig. 1 and Fig. 2.
Materials
- Proteinase K (Boehringer Mannheim Biochemicals, Germany
or Sigma Chemical Co., U.S.A)
Correspondence to Shigeyoshi Oba, The University of Tokyo, The 2nd Department ofInternal Medicine, Faculty of Medicine, 3-7-1 Hongo, Bunkyoku, Tokyo, Japan (phone +81-3-3815-5411 (ext. 3056);fax +81-3-3814-0021;
e-mail oba-2im@h.u-tokyo.ac.jp); Takehiko Koji, Nagasaki University,
Dept. of Histology and Cell Biology, 1-12-4, Sakamoto, Nagasaki, 8528523, Japan
In Situ Nick End-labeling: light Microscopical
SHIGEYOSHI OBA AND TAKEHIKO KOJI
Introduction
It has been postulated that in apoptotic cell death, the first biochemical event is single-strand DNA breaks followed by doublestrand DNA cleavage in the linker regions between nucleosomes
(Peitsh 1993). TUNEL (Terminal transferase-mediated dUTPbiotin nick end labeling) is a convenient molecular histochemical tool to detect these single-DN A breaks. Briefly described, this
tool is based on repeated addition of biotin-dUTP at the 3-hydroxy ends of DNA of tissue by terminal deoxynucleotidyl transferase and detection of these additional biotin-dUTP by HRP labeled anti-biotin antibody. You can use digoxigenin-dUTP or
fluorescent conjugated dUTP instead of biotin-dUTP.
Outline
The entire protocol of TUNEL is outlined in Fig. 1 and Fig. 2.
Materials
- Proteinase K (Boehringer Mannheim Biochemicals, Germany
or Sigma Chemical Co., U.S.A)
Correspondence to Shigeyoshi Oba, The University of Tokyo, The 2nd Department ofInternal Medicine, Faculty of Medicine, 3-7-1 Hongo, Bunkyoku, Tokyo, Japan (phone +81-3-3815-5411 (ext. 3056);fax +81-3-3814-0021;
e-mail oba-2im@h.u-tokyo.ac.jp); Takehiko Koji, Nagasaki University,
Dept. of Histology and Cell Biology, 1-12-4, Sakamoto, Nagasaki, 8528523, Japan