Sirius red and acid fuchsin staining mechanisms

The purpose of the study was to investigate the staining mechanism of acid fuchsin and Sirius red. Acid (poly-glutamic acid), neutral (poly-hydroxyproline) and basic (poly-arginine, poly-histidine, poly-lysine) poly-amino acids, collagen types I, II and III, and arginine- and lysine-containing histones were used as test substances applied to nitrocellulose membranes as dot blots. Five micrometer sections of granulation tissue on slides were tested in parallel. Some dots and sections were treated with chloramine-T before staining with acid fuchsin and Sirius red and some with 1 M NaOH after staining. The acid and neutral poly-amino acids were not stained, but the basic amino acids polylysine and poly-arginine, poly-amino acids containing these basic amino acids and the histones and the collagens exhibited intense staining. Oxidative deamination by chloramine-T abolished the staining and 1 M NaOH removed the staining except in the case of poly-arginine. Tissue sections treated in the same way showed a considerable decrease in staining after oxidative deamination with chloramine-T; in particular, the staining of the smaller fibers was abolished. The staining was totally removed by destaining with 1 M NaOH. Therefore, acid fuchsin and Sirius red are not selectively bound to collagen; they are also bound to other proteins containing basic amino acids, and staining to a large extent is influenced by electrostatic forces. The staining seems not to be selective for collagen, and one must account for this when quantitative conclusions are drawn from collagen methods using these stains.