A Melanin bleaching method to prevent non-specific immunostaining of chicken feathers

Abstract

Melanin in pigmented organs like the skin is known to react with 3,3′-diaminobenzidine (DAB) to give a brown colour indistinguishable from the colour that DAB imparts to target antibodies bound to specific antigens. This can lead to false positives in chicken feathers during immunoperoxidase staining. Here, we present a simple, fast and practical method for bleaching chicken feathers which can be applied prior to immunohistochemistry staining without affecting specific antigen-antibody binding. To our knowledge, this is the first report of a melanin-bleaching technique prior to immunoperoxidase staining techniques of chicken feathers for detection of pathogens. Optimisations of the method include: <br/> • Removal of melanin from tissue sections using a short incubation with potassium permanganate followed by incubation with oxalic acid prior to immunostaining for improved specificity. <br/> • This technique did not affect the antigenicity of infectious laryngotracheitis virus antigen and did not cause damage or detachment of tissues from the slides.