A combination Prussian blue – hematoxylin and eosin staining technique for identification of iron and other histological features

Abstract

The Prussian blue reaction (PB) detects ferric iron in histological sections but the nuclear fast red (NFR) counterstain does not selectively stain the surrounding tissue and cellular features very well. The PB/NFR stain has the advantage of detecting iron located in tissue sections, but a significant disadvantage of having poorly differentiated tissue components, as compared to a routine hematoxylin and eosin (H&E). We developed a combination of Gomori’s Prussian blue/H&E staining method (PB/H&E), and modified the technique for best performance and clarity, then assessed the ability of this new combination stain to differentiate histological features of the tissue and identify iron. Serial sections from seven formalin fixed paraffin-embedded liver samples previously diagnosed with the presence of ferric iron were subjected to our routine H&E, routine PB/NFR and three trials of the new Prussian blue/H&E combination (PB/H&E). The technique that best differentiated the histological components of tissues containing iron was further tested on liver sections from a variety of species to verify consistency i.e. equivalence in staining intensity, concentration, brightness between sections of the same sample and quality i.e. coloration, vividness, recognizable differentiation of tissue components, improved staining.