Histochemical Demonstration of DNA Double Strand Breaks by in Situ 3′-tailing Reaction in Apoptotic Endometrium

Abstract

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 λ-exonuclease (λEx) prior to the TdT reaction to generate 3′-protruding ends. The λEx-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.

Key Words:

• DNA double strand breaks
• apoptosis
• 3′-tailing reaction
• terminal deoxynucleotidyl transferase
• λ-exonuclease
• histochemistry