I'm questioning if it is necessary to include a negative control (incubated with label mix solution in absence of TdT) in a TUNEL assay for evaluation of DNA fragmentation? Will dUTP label to 3'-OH of DNA nick spontaneously without TdT? I have observed several weak FITC fluorescent in the negative control slide under microscopy; however, the intensity of FITC is higher in the test sample (TdT+ label mix) than in negative control. Thanks.
If you are not certain, I think you should include the TdT-free control, if only to be comfortable with your own result.
Hi Rafael Linden ,
Thanks for your response. I have included a TdT-free control in my TUNEL experiment. Many weak FITC+ cells were observed in the TdT-free control slide under microscopy by using BioVision ApoDIRECT In situ DNA Fragmentation assay ki (CN.: K402-50) or Roche TUNEL label mix (CN.: 11767291910) plus a TdT enzyme. However, no FITC+ cells were observed in TdT-free control slide when Roche In situ cell death detection kit (CN.: 11684795910) was used. I was wondering whether the cause of false-positive results was due to inappropriate ratio of FITC-dUTP and unlabeled dNTP. Thx.
Hello, Just a quick question, so for negative control, no cells must be stained?
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