Congo Red and amyloids: history and relationship

Abstract

Staining with Congo Red (CR) is a qualitative method used for the identification of amyloids in vitro and in tissue sections. However, the drawbacks and artefacts obtained when using this dye can be found both in vitro and in vivo Analysis of scientific data from previous studies shows that CR staining alone is not sufficient for confirmation of the amyloid nature of protein aggregates in vitro or for diagnosis of amyloidosis in tissue sections. In the present paper, we describe the characteristics and limitations of other methods used for amyloid studies. Our historical review on the use of CR staining for amyloid studies may provide insight into the pitfalls and caveats related to this technique for researchers considering using this dye.

Keywords: Congo red; amyloid detection; amyloid dye; amyloid staining; amyloidosis; amyloids.

Figure 1. The main hypothetical models of the binding of CR to amyloids

(A) Dye binding mediated by hydrogen bonding between primary hydroxyl groups of the peptide chain (similar to the polysaccharide chain) and the amino groups of CR (Puchtler et al., 1962 [132]). (B) CR molecule could bind to positively charged amino acid residues along the peptide chains (Klunk, W.E. et al., 1989 [161]). (C) Polar contacts drive CR binding (Reinke and Gestwicki, 2011 [166]).

Figure 2. Investigation of SMT aggregates

(A) SMT(KCl) aggregates: X-ray diffraction (left); CR polarisation microscopy of the aggregates, scale: 1 μm (top right); electron microscopy of negatively stained aggregates, scale: 100 nm (bottom right). (B) SMT(Gly) aggregates: CR polarisation microscopy of the aggregates, scale: 1 μm (top left); electron microscopy of negatively stained aggregates, scale: 100 nm (bottom left); X-ray diffraction (right). (C) SMT(KCl) aggregates after partial disaggregation: X-ray diffraction (left), electron microscopy of negatively stained titin aggregates, scale: 100 nm (right). (D) SMT(Gly) aggregates after partial disaggregation: electron microscopy of negatively stained aggregates, scale: 100 nm (left); X-ray diffraction (right). (E) Microscopy under polarised light of a dried drop of buffer containing 0.15 M glycine-KOH, pH 7.2–7.4 and CR, scale: 100 μm. For electron microscopy, 2% aqueous uranyl acetate staining was used.