30
SHIGEYOSHI OBA AND TAKEHIKO KOTI
Results
This method can detect the apoptotic cells without condensed
nuclei in situ. For examples, apoptotic cells of ischemia-reperfusion kidneys were shown Fig. 3 and Fig. 4. Fig. 3 shows that the
samples treated with lO)lg/ml Proteinase K are reacted with
terminal deoxynucleotidyl transferase reasonably, although
those treated with 1.0)lg/ml Proteinase K are not reacted well
with TdT. Fig. 4 shows that the samples treated with TdT reaction
solution containing 0.25)lM biotin-16-dUTP stain moderately
(Fig. 4 a), although there is excessive background staining in
the samples treated with 1,2.5, 5)lM biotin-16-dUTP(Fig 4 b,c,d).
Troubleshooting
• Care must be taken not to dry up samples, when you tap off
excess buffer and wipe around specimens. Drying up was
main cause of non specific reaction.
• You must treat negative controls without TdT.
• You must treat TdT carefully because it may easily lose activity below or up -20°C.
Comments
This method can detect apoptosis cell at early phase but it can not
discriminate between apoptosis and necrosis. You must ascertain the existence of apoptosis by using another method, for example, formation of DNA lad dering, electron microscoptic observation, F ACS.
References
Adams Je.; J. Histochem Cytochem 29: 775, 1981
Gavieli, Y., Sherman, Y., Ben-Sasson, S.A.; J. Cell Biol., 119:493-501,1992
Peitsh MC, Muller C, Tschopp J (1993) DNA fragmentation during apoptosis is caused by frequent single-strand cuts. Nucl. Acid Res. 21:42064209
SHIGEYOSHI OBA AND TAKEHIKO KOTI
Results
This method can detect the apoptotic cells without condensed
nuclei in situ. For examples, apoptotic cells of ischemia-reperfusion kidneys were shown Fig. 3 and Fig. 4. Fig. 3 shows that the
samples treated with lO)lg/ml Proteinase K are reacted with
terminal deoxynucleotidyl transferase reasonably, although
those treated with 1.0)lg/ml Proteinase K are not reacted well
with TdT. Fig. 4 shows that the samples treated with TdT reaction
solution containing 0.25)lM biotin-16-dUTP stain moderately
(Fig. 4 a), although there is excessive background staining in
the samples treated with 1,2.5, 5)lM biotin-16-dUTP(Fig 4 b,c,d).
Troubleshooting
• Care must be taken not to dry up samples, when you tap off
excess buffer and wipe around specimens. Drying up was
main cause of non specific reaction.
• You must treat negative controls without TdT.
• You must treat TdT carefully because it may easily lose activity below or up -20°C.
Comments
This method can detect apoptosis cell at early phase but it can not
discriminate between apoptosis and necrosis. You must ascertain the existence of apoptosis by using another method, for example, formation of DNA lad dering, electron microscoptic observation, F ACS.
References
Adams Je.; J. Histochem Cytochem 29: 775, 1981
Gavieli, Y., Sherman, Y., Ben-Sasson, S.A.; J. Cell Biol., 119:493-501,1992
Peitsh MC, Muller C, Tschopp J (1993) DNA fragmentation during apoptosis is caused by frequent single-strand cuts. Nucl. Acid Res. 21:42064209