2 In Situ Nick Translation
15
- Fresh frozen samples: Tissues are embedded in OCT com- Samples
pound (Miles, USA), cut(5-6).lm), placed onto silane coated
glass slides and air dried. Then sections are fixed with ethanol/acetic acid (3:1, v/v) for 20 mins, and washed 3 times with
PBS for 5 mins each time.
- Formalin-fixed and paraffin embedded samples: Tissues are
cut (4 ).lm), placed onto silane-coated glass slides and deparaffined.
- Proteinase K (PK) (Sigma, USA)
- Biotin-ll-dUTP (ENZO Diagnostics, Inc., USA)
- E.coli DNA polymerase I (Takara Co., Japan)
- Horseradish peroxydase (HRP) labeled anti-biotin antibody
(Vector Lab. Inc., USA)
NT buffer:
- 50 mM Tris/HCI (pH 7.5)
10 mM MgCl2
0.1 mM dithiothreitol
50 ).lg/ml BSA
20).lM dATP, 20).lM dGTP and 20).lM dCTP
20 ).lM TTP(negative control) or 20 ).lM biotin-ll-dUTP
Chromogenic solution (Adams 1981):
- 0.1 M sodium phosphate buffer (pH 7.5)
- 0.5 mg/ml 3-3' -diaminobenzidine/4HCI
- 0.01% hydrogen peroxide
- 0.025% CoCh
- 0.02% NiS04(NH4hS04
Procedure
1. Immerse in PBS.
2. If necessary, PK digestion for 15 mins at 37°C and washing in
PBS.
(Conc. of PK is recommended at 1 ).lg/ml for fresh frozen
sample and 10 ).lg/ml for paraffin embedded samples.)
3. Keep in 50 mM Tris/HCl.
4. Nick translation at 37°C for 3 hrs in a moist chamber
Reaction with E.coli DNA polymerase 1(200 U/ml) in NT
buffer.