Congo Red and Iodine-Stained Amyloid Fibrils Studied by Negative Contrast Electron Microscopy and X-Ray Diffraction

The compound formed by addition of Congo red to suspensions of amyloid fibrils is known to be specific for amyloid and fairly stable. It is analogous to the addition or inclusion compounds of Congo red with cellulose. At one time it was suggested that the Congo red molecule with dimensions of 21 x 8.5 x 3.8 X is sequestered within the fibril. Ashkenazi and his colleagues have demonstrated that the Congo red amyloid complex forms only when the amyloid fibril has its normal sugar content, and, that the complex resists alkalai that would ordinarily remove the sugar content. The conclusion was that Congo red stains the carbohydrate moiety of amyloid. 1% aqueous Congo red was added slowly to relatively pure human liver amyloid fibril suspensions prepared from successive aqueous centri-fugations at 4,000 G of upper precipitate layers. Addition was stopped when the supernatant after centrifugation was reddish. The red precipitate was washed for 9 minutes twice in distilled water, and studied by electron microscopy before and after the addition of equal parts of 2% pH 6 potassium phosphotungstate. Amyloid controls free of Congo red were also studied. (RCA EMU 2A; carbon-coated “Formvar” grids).