Cell Biology

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).

Plants have developed two distinct mechanisms, i.e., strategy I (reduction strategy) and II (chelation strategy), to mobilize insoluble Fe(III) in the rhizosphere and transport it through the plasma membrane. Arabidopsis thaliana and other dicots rely on strategy I. In this strategy, the rhizosphere is first acidified by a PM-localized H+-ATPase, AHA2. Then, FERRIC CHELATE REDUCTASE 2 (FRO2) reduces Fe(III) to soluble Fe(II). Finally, the reduced Fe is taken up by a high-affinity transporter, IRON-REGULATED TRANSPORTER 1 (IRT1). Root ferric chelate reductase activity can be quantified spectrophotometrically by the formation of Purple-colored Fe(II)-ferrozine complex in darkness.

Purpose: To demonstrate ferric iron in tissue sections. Small amounts of iron are found normally in spleen and bone marrow. Excessive amounts are present in hemochromatosis, with deposits found in the liver and pancreas, hemosiderosis, with deposits in the liver, spleen, and lymph nodes.

Principle: The reaction occurs with the treatment of sections in acid solutions of ferrocyanides. Any ferric ion (+3) in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called 'Prussian blue" or ferric ferrocyanide.