Nazir Shahid, Charlesworth Richard P G, Walkden-Brown Stephen W, Gerber Priscilla F
Animal Science, School of Environmental and Rural Science, University of New England, Australia.
School of Science and Technology, University of New England, Australia.
MethodsX. 2020 Jun 11;7:100957. doi: 10.1016/j.mex.2020.100957. eCollection 2020.
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:•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.•This technique did not affect the antigenicity of infectious laryngotracheitis virus antigen and did not cause damage or detachment of tissues from the slides.
已知皮肤等色素沉着器官中的黑色素会与3,3'-二氨基联苯胺（DAB）发生反应，产生一种棕色，这种棕色与DAB赋予与特定抗原结合的靶抗体的颜色无法区分。这可能会在免疫过氧化物酶染色过程中导致鸡毛出现假阳性。在此，我们提出一种简单、快速且实用的鸡毛漂白方法，该方法可在免疫组织化学染色之前应用，且不会影响特异性抗原 - 抗体结合。据我们所知，这是关于在鸡毛免疫过氧化物酶染色技术之前用于检测病原体的黑色素漂白技术的首次报告。该方法的优化包括：
• 在免疫染色前，先用高锰酸钾短暂孵育组织切片以去除黑色素，然后用草酸孵育，以提高特异性。
• 该技术不影响传染性喉气管炎病毒抗原的抗原性，也不会导致组织从载玻片上损伤或脱落。
