16
SATOSHI HASHIMOTO AND TAKASHI KANEMATSU
5. Wash 3 times with 50 mM Tris/HCI for 5 mins, each time and
rinse once with PBS.
6. Blocking with 500 ).lg/ml normal goat IgG and 5% BSA/PBS
at RT for 1 hr.
7. Reaction with HRP-goat anti Biotin for 3 hrs.
8. Wash with 0.075% Brij.lPBS for 15 mins, 4 times.
9. Visualize the sites ofHRP by immersing in chromogenic solution for 5-6 mins.
10. Dehydration with serial upgraded ethanol solutions, clear
with xylene and mount without counterstaining.
Results
Nicks are visualized as intense signal in nuclei (Fig. 2 a,b,c). Signal is varied from fixation or proteinase treatment of samples
(Koji 1996){Hashimoto 1995). As a negative control, some secI
I.
\
I .;
apoplptlc body.
(phagocytized)
Fig. 2. In situ nick translation in human colorectal cancer sections. a Some nuclei of normal appearance (N) and apoptotic bodies phagocytized by a neighbouring cancer cell were stained. b Mitotic cells (M) were not stained. c Distribution of nuclei labeled with ISNT.
SATOSHI HASHIMOTO AND TAKASHI KANEMATSU
5. Wash 3 times with 50 mM Tris/HCI for 5 mins, each time and
rinse once with PBS.
6. Blocking with 500 ).lg/ml normal goat IgG and 5% BSA/PBS
at RT for 1 hr.
7. Reaction with HRP-goat anti Biotin for 3 hrs.
8. Wash with 0.075% Brij.lPBS for 15 mins, 4 times.
9. Visualize the sites ofHRP by immersing in chromogenic solution for 5-6 mins.
10. Dehydration with serial upgraded ethanol solutions, clear
with xylene and mount without counterstaining.
Results
Nicks are visualized as intense signal in nuclei (Fig. 2 a,b,c). Signal is varied from fixation or proteinase treatment of samples
(Koji 1996){Hashimoto 1995). As a negative control, some secI
I.
\
I .;
apoplptlc body.
(phagocytized)
Fig. 2. In situ nick translation in human colorectal cancer sections. a Some nuclei of normal appearance (N) and apoptotic bodies phagocytized by a neighbouring cancer cell were stained. b Mitotic cells (M) were not stained. c Distribution of nuclei labeled with ISNT.