Evaluation of the effects of melanin bleaching in different steps of immunohistochemistry on an automated platform

Melanin is a granular brown-black pigment unevenly distributed in tissues. Severe melanin deposition can obscure cellular morphological features and tissue structures, hindering the evaluation of melanocytic lesion. This condition is particularly prone to interfering with the evaluation of immunohistochemistry (IHC), thereby affecting diagnostic accuracy. Therefore, melanin bleaching techniques have become a crucial step, enabling pathologists to better examine melanin-rich tissue specimens. In this study, we utilized low-concentration 1 % hydrogen peroxide for melanin bleaching and integrated the bleaching process into IHC on the same platform to establish a continuous workflow. We compared melanin bleaching in different steps to see the effect of bleaching on IHC. Three different bleaching protocols were designed: bleaching (i) before antigen retrieval, (ii) after antigen retrieval, and (iii) after IHC staining. The effects of melanin bleaching on tissue morphology preservation and IHC quality in heavily pigmented melanocytic lesions were evaluated. The results showed that melanin bleaching before antigen retrieval ensured accurate localization of expression of HMB45, Melan A, and SOX10. This method showed no background staining, preserved tissue morphology without detachment, and maintained antigen immunogenicity. The pre-retrieval bleaching protocol allowed for clearer IHC staining result and can be applied on automated platform and routine staining workflows.