Dear all,
is anyone of you experienced how to improve the reproducibility of TUNEL assay in plant/moss cell culture? I am trying to establish this assay to visualize DNA damages in nuclei.
When I compare different biological replicates and samples, the variation is very high. Because of the high variation of nuclei with TUNEL-signal (Fluorescein) of all nuclei (DAPI) between different biological replicates (same line, standardized growth conditions in liquid culture, no changes in the protocol or chemicals used) the setup obviously needs improvement, has anyone suggestions? I always include a negative control (no TdT enzyme) and a positive control (DNaseI digested sample) which look fine.
I start with fixation for 3h, followed by removal of chlorophyll by a EtOH series (50%, 70%, 96%), 2 times wash with PBS. Permeabilization is done overnight, wash in PBS once, the TUNEL reaction using Fluorescein-12-dUTP and TdT is incubated for 1h30, afterwards the samples are washed twice in DAPI solution.
Thanks a lot for any suggestions!
Gertrud
Hi Gertrud,
Maybe specific pre-tretments as per attached link may help reduce the false positive staining. Hopefully you can translate it to your tissue sample.
http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC395637&blobtype=pdf
Dear Dr. Denise M. Barbulescu ,
Thanks for the reference it is useful to me also.
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