2 In Situ Nick Translation
17
tions in the absence of DNA polymerase and/or biotinylated
dUTP which inform specificity of the reaction should be added
and their signals are essentially blank.
Troubleshooting
Heavy proteinase treatment results in a high background staining and a destruction of the samples especially in their edge.
First, the optimal conc. of PK is determined according to the
fixation or organ of the tissues respectively. And PK digestion
should be performed at 37°C not at RT.
Comments
We have applied this technique to characterize human colorectal
cancers (Hashimoto 1997). In paraffin sections, nuclei labeled
with ISNT were different from Proliferating cell nuclear antigen
positive nuclei. DNA obtained from cancer tissues that contained many ISNT positive nuclei electrophoresed as a ladder
pattern. Nuclei labeled with ISNT coincide with the morphologic
apoptosis and hallmark of apoptosis.
And in frozen sections, ISNT is a convenient molecular histochemical tool not only to detect cell death but also to discriminate both types of cell death, apoptosis and necrosis (Hashimoto 1995).
References
Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction
product. J Histochem Cytochem 29: 775
Appleby DW, Modak SP (1977) DNA degradation in terminally differentiating lens fiber from chick embroys. Proc Natl Acad Sci USA 74:
5579-5583
Dawson BA, Lough J (1988) Immunocytochemical localization of transient
DNA strand breaks in differentiating myotubes using in situ nick-translation. Dev BioI 127:362-367
Hashimoto S, Koji T, Niu J, Kanematsu T, Nakane PK (1995) Differential
staining of DNA strand breaks in dying cells by non-radioactive in situ
nick translation. Arch Histol Cytol 58:161-170
17
tions in the absence of DNA polymerase and/or biotinylated
dUTP which inform specificity of the reaction should be added
and their signals are essentially blank.
Troubleshooting
Heavy proteinase treatment results in a high background staining and a destruction of the samples especially in their edge.
First, the optimal conc. of PK is determined according to the
fixation or organ of the tissues respectively. And PK digestion
should be performed at 37°C not at RT.
Comments
We have applied this technique to characterize human colorectal
cancers (Hashimoto 1997). In paraffin sections, nuclei labeled
with ISNT were different from Proliferating cell nuclear antigen
positive nuclei. DNA obtained from cancer tissues that contained many ISNT positive nuclei electrophoresed as a ladder
pattern. Nuclei labeled with ISNT coincide with the morphologic
apoptosis and hallmark of apoptosis.
And in frozen sections, ISNT is a convenient molecular histochemical tool not only to detect cell death but also to discriminate both types of cell death, apoptosis and necrosis (Hashimoto 1995).
References
Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction
product. J Histochem Cytochem 29: 775
Appleby DW, Modak SP (1977) DNA degradation in terminally differentiating lens fiber from chick embroys. Proc Natl Acad Sci USA 74:
5579-5583
Dawson BA, Lough J (1988) Immunocytochemical localization of transient
DNA strand breaks in differentiating myotubes using in situ nick-translation. Dev BioI 127:362-367
Hashimoto S, Koji T, Niu J, Kanematsu T, Nakane PK (1995) Differential
staining of DNA strand breaks in dying cells by non-radioactive in situ
nick translation. Arch Histol Cytol 58:161-170