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 positivity by pretreatment with single-stranded DNA digestion enzymes, S1 nuclease, and mung bean nuclease. When mung bean nuclease (5 U/50 microliter/section) was incubated at 37 degrees C for 30 min, the TUNEL reaction was most effectively enhanced. Pretreatment with S1 nuclease (0.25 U/50 microliter/section) at 37 degrees C for 45 min was less reliable. Compared with the conventional TUNEL sequence, the enhancement technique using mung bean nuclease enabled us to detect more apoptotic cells in human small intestine, colon, tonsil, thymus, endometrium, ovary, liver, kidney, and pancreas. The positivity was not affected by autolytic change. The mechanism of enhancement is discussed.
Umemura
S
S
Department of Pathology, Tokai University School of Medicine, Isehara, Japan.
Yasuda
M
M
Osamura
R Y
RY
Kawarada
Y
Y
Sugiyama
T
T
Tsutsumi
Y
Y
D017209:Apoptosis; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000276:immunology / Q000378:metabolism*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D007887:Fabaceae / Q000201:enzymology; D006801:Humans; D007124:Immunoenzyme Techniques; D010946:Plants, Medicinal; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D016707:Tissue Fixation
D004277:DNA, Single-Stranded; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D001710:Biotin; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D003851:Deoxyribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases