ENHANCEMENT OF TDT-MEDIATED DUTP-BIOTIN NICK END-LABELING (TUNEL) METHOD USING MUNG BEAN NUCLEASE, A SINGLE-STRANDED-DNA DIGESTION ENZYME

The TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method has been employed widely to demonstrate apoptotic cells in routinely prepared paraffin sections. Because the apoptotic cells were reactive with the antibody to single-stranded DNA, we attempted to enhance the TUNEL pos itivity by pretreatment with single-stranded DNA digestion enzymes, S1 nuclease, and mung bean nuclease. When mung bean nuclease (5 U/50 mu l/section) was incubated at 37 degrees C for 30 min, the TUNEL reactio n was most effectively enhanced. Pretreatment with S1 nuclease (0.25 U /50 mu l/section) at 37 degrees C for 45 min was less reliable. Compar ed with the conventional TUNEL sequence, the enhancement technique usi ng mung bean nuclease enabled us to detect more apoptotic cells in hum an small intestine, colon, tonsil, thymus, endometrium, ovary, liver, kidney, and pancreas. The positivity was not affected by autolytic cha nge. The mechanism of enhancement is discussed.