Histochemical demonstration of DNA double strand breaks by in situ 3'-tailing reaction in apoptotic endometrium.
A new histochemical technique, called in situ 3'-tailing reaction (ISTR), to detect DNA double strand breaks (DSB) was developed and applied to tissue sections of apoptotic endometrium. To demonstrate DSB, biotin-labeled and unlabeled dATPs with terminal deoxynucleotidyl transferase (TdT) were added to the many 3-hydroxyl termini of DNA fragments generated in the apoptotic cells. For an efficient 3'-end labeling, it was necessary to treat the sections with lambda-exonuclease (lambdaEx) prior to the TdT reaction to generate 3'-protruding ends. The lambdaEx-TdT reaction specifically labeled nuclear fragments in the apoptotic cells in paraformaldehyde fixed frozen sections. In paraffin sections, pretreatment with proteinase K was effective for 3'-tailing reaction. ISTR should be a useful tool for detecting dying cells in both physiological and pathological states.
Nakamura
T
T
Department of Pathology, Shinshu University School of Medicine, Matsumoto, Japan.
Sakai
T
T
Hotchi
M
M
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D003838:Deoxyadenine Nucleotides; D004587:Electrophoresis, Agar Gel; D004717:Endometrium / Q000737:chemistry* / Q000166:cytology; D019286:Endopeptidase K; D004801:Eosine Yellowish-(YS); D005090:Exodeoxyribonucleases; D005260:Female; D006416:Hematoxylin; D006651:Histocytochemistry / Q000379:methods*; D011817:Rabbits; D012697:Serine Endopeptidases / Q000378:metabolism; D014764:Viral Proteins
D003838:Deoxyadenine Nucleotides; D014764:Viral Proteins; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D004801:Eosine Yellowish-(YS); D006416:Hematoxylin