Dear DaudiMy name is Kelly Avila. I´m doing my PhD in Biotechnology...

Hi Kelly,
Thanks for your message. Could you share some representative images of your leaves using the "Reproducibility Feedback" feature above? That way I can see how the staining is spread across your leaves and advise you on the best programs/tools for quantifying the stain.

Hi Arsalan

Thanks a lot for yor your help.

Taking account all your advices and the other questions. I wondering if is better quantify intensity of staining or just express as positive or negative. Because the stain is not uniform. I tried with different programs but quantifiy this kind of stains is difficult. Any suggestion?

Hi Kelly, you are right about the chemistry: DAB is oxidized by hydrogen peroxide, which in turn leads to the dark brown precipitate/color you see. Hydrogen peroxide is not necessarily only produced by pathogen attack (i.e. the oxidative burst) - This is where you may be having a problem. ROS is produced due to all kinds of stresses on plant tissue - so if your samples are stressed for any reason, there may be background ROS (and hence the brown stain). The key is to minimize any other stress so the hydrogen peroxide produced by the pathogen attack is relatively higher than basal levels, and this difference is observed by staining. Does this make sense?

Dear Daudi,


I'm still working with this protocol. But I had problems with the results, I think that these results is for technical details.
But I wondering why in DAB protocols is don´t required extra H2O2 like other protocols.
I would like to confirm (maybe I´m confused with the chemistry of reaction).
If DAB is oxided by H2O2, the ending of the test is quantify indirectly H2O2 production, and I can assume that brown coloration is in response to pathogen attack, so all brown stains are produced by the pathogen presence

Hi Kelly, thanks for your message. Hope I can help. Using leaf discs is actually a more common way to do DAB (and most types of ROS) staining. I just did whole leaves because it is easy with Arabidopsis as they're small, similar sized and easy to fit into 12 and 6 well plates. I would encourage you to use leaf discs for you oil palm leaves. The bigger the better, so 0.5 cm diameter minimum I would suggest as I guess your leaves are quite big. There will be mechanical stress when you cut the leaves (ideally with a cork borer), but the way to overcome this is to allow your leaf discs to equilibrate and stabilize in sterile water for about 12 - 24 hours (without any agitation). Then you can proceed to do the DAB assay on those discs as described above. Also make a note of the new DAB I recommend to use (in one of the comments above). Please update us with your results and how it worked in your hands (on this same Q&A thread). Good luck!!