3 In Situ Nick End-Labeling: Light Microscopical
23
TdT reaction solution (total 10001l1)-digoxigenin
distilled water
5X reaction buffer (Boehringer Industries)
DTT 10mM
CoC12 25mM (Boehringer Industries)
701).d
200111
10111 (O.lmM)
60111 (1.5mM)
dATP ImM
20).11 (20IlM)
digoxigenin-ll-dUTP (Boehringer Industries,lmM) 5).11 (5).1M)
TdT (Boehringer Industries, 25U/ml)
Terminal transferase kit (Boehringer Mannheim Biochemicals, Germany;
No. 220582) contains 5X reaction buffer, CoC12 and TdT
3,3' -diaminobenzidine(DAB) reaction solution
distilled water
90 ml
DAB
50 mg
1 M phosphate buffer, pH 1.2
10 ml
CoC12 (1 %)
2.5 ml
NiS04 (1 %)
2.0 ml
30% hydrogen peroxide (H20 2)
33).11
In our protocol, the sites of horseradish peroxides(HRP) were
visualized with hydrogen peroxide and 3,3' -diamnobenzidine
(DAB) in the presence of nickel and cobalt irons, which increase
the sensitivity more than tenfold as compared to that of hydrogen peroxide and DAB alone (Adams 1981).