A sensitive post-DAB enhancement technique for demonstration of iron in the central nervous system

A technique is described for enhancing the reaction product of the staining reaction for iron in paraffin-embedded tissue from central nervous system (CNS). After amplification of the Prussian Blue staining reaction with 3,3'-diaminobenzidine (DAB), the reaction product was further intensified using a stepwise treatment with silver methenamine, gold chloride and uranyl nitrate (post-DAB treatment). Following the Prussian Blue-DAB staining reaction, iron was seen only in glial cells and choroid plexus epithelial cells, whereas the post-DAB treatment revealed that neurons and endothelial cells of the brain capillaries were also positively stained. The post-DAB treatment resulted additionally in an increased intensity of the reaction product within choroid plexus epithelial cells compared to that obtained in sections subjected only to the Prussian Blue-DAB reaction. The reliability of the method was evaluated using liver sections as positive controls. Furthermore the higher sensitivity of the method was assessed using nitrocellulose filters containing serially diluted iron-saturated transferrin. The post-DAB method is simple and can easily be applied to formalin- or glutaraldehyde fixed, paraffin-embedded nervous and non-nervous tissue.