Abstract
Visualization of iron (Fe) localization in plants has greatly enhanced our understanding of plant Fe homeostasis. One of the relatively simple and yet powerful techniques is the classical Perls blue stain (Perls, 1867). The technique is based on the conversion of ferrocyanide to insoluble crystals of Prussian blue in the presence of Fe3+ under acidic conditions. It has been extensively used in animal and human histology (Meguro et al., 2007) and has recently gained popularity in plant research. For specific purposes, Fe signals may be additionally enhanced in the 3,3’-diaminobenzidine tetrahydrochloride (DAB) intensification procedure (Meguro et al., 2007). It has been demonstrated that this intensification results in the detection of both Fe2+ and Fe3+ ions (Roschzttardtz et al., 2009). The method has been successfully applied at the whole plant, organ and subcellular levels, both with (Roschzttardtz et al., 2011; Schuler et al., 2012; Roschzttardtz et al., 2013; Ivanov et al., 2014) and without intensification (Stacey et al., 2008; Long et al., 2010).Here, we present a full Perls staining and DAB intensification protocol, the way it is performed in our lab (Ivanov et al., 2014).
Keywords: Perls staining, DAB intensification, Iron deficiency, Plant
Materials and Reagents
Equipment
Procedure
Notes
Representative data
Figure 1. Visualization of Fe in Arabidopsis root by Perls staining and DAB intensification. A. A fixed 7 day-old Arabidopsis seedling. B. Perls-stained root. Signal is seen in the apoplast and central cylinder. C. DAB intensification applied without prior Perls staining. No specific signal can be seen. D. Perls-DAB staining on a root, grown on agar plate under sufficient Fe supply. Strong signal can be seen in the apoplast. E. Perls-DAB staining on a root, grown on agar plate under sufficient Fe supply and then transferred for 24 to Fe-deficient medium. Staining can be seen in the central cylinder. Low to no signal is present at the apoplast. The zone represented on the closeup images corresponds to the highlighted zone in A. Bars represent 1 mm.
Recipes
Acknowledgments
This protocol is based on the procedure described by Roschzttardtz et al. (2009). Research in the authors’ laboratory was supported by the Saarland University, Germany, and the Heinrich Heine University, Germany. We would like to thank Ailisa Blum for helping introduce the technique in our lab.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Dear Paula,Recently, we had cases of blue staining remaining in the base of the root (next to the hypocotyl). This happened with plants that take up more iron and the overall iron content was strongly elevated.I suppose the fixation and infiltration conditions might have to be optimized in this case but we have not tackled the problem yet. I will write back if we find a solution for this one.Rumen
Dear Paula,thanks for the information. We have never used unfixed material for the procedure. To my knowledge, the problem lies with DAB, since in living tissues it would react with the endogenously generated hydrogen peroxide. This might be a problem when the plant is heavily stressed in the steps preceding the DAB addition.For the potassium phosphate buffer, we did not use that either but my guess is that it should work the same way the sodium buffer.Best wishes,Rumen