Abstract
Plant cells continually produce reactive oxygen species (ROS) as a by-product of aerobic metabolism. Increased production of ROS occurs under unfavorable conditions imposed by various abiotic and biotic factors. Accumulation of ROS is damaging to various cellular components and macromolecules including plasma membrane, nucleic acids, and proteins and eventually leads to cell death. In this protocol, we describe the histochemical detection of hydrogen peroxide (H2O2) and superoxide (O2-) anion, two of the most important ROS, in Brassica juncea seedlings by using 3,3ʹ-Diaminobenzidine (DAB) and Nitrotetrazolium blue chloride (NBT) as the chromogenic substrate. DAB is oxidized by H2O2 in the presence of peroxidases and produces reddish brown precipitate. NBT reacts with O2- to form a dark blue insoluble formazan compound. The protocol can be used in other plant species and for different plant tissues.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
The protocol was implemented in a project that was partially funded by the Council of Scientific and Industrial Research (CSIR) Grant No. 38/1126/EMR-II. DK and PS acknowledge the financial support from CSIR and University Grants Commission (UGC). MAY was a UGC-Dr. D.S. Kothari Postdoctoral Fellow during the course of this project. Research in the laboratory of NBS is supported by U.G.C.-C.A.S., U.G.C.-R.N.W., Department of Science and Technology (D.S.T.)-F.I.S.T., and D.S.T.-PURSE. The authors would like to acknowledge Thordal-Christensen et al. (1997) and Jabs et al. (1996) (kindly see the reference section) whose work was adapted in the present protocol.
References
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NBT dissolves well, DAB. I stirred it with magnetic suspension light all night, but it didn't dissolve... In the end, it can only become turbid.. But it can be dyed.... The picture obtained by the above method is not very good, it is suggested to see the original literature of this method.. It's very detailed....