Depigmentation of Melanin-containing Tissues Using Hypochlorous Acid to Enhance Hematoxylin-eosin and Immunohistochemical Staining

Abstract

Pathologists diagnose diseases by observing the histologic and cellular morphology microscopically. However, the high pigmentation in melanin-containing tumors can hide the tumor cell structures, making diagnosing challenging. Previously, hydrogen peroxide and potassium permanganate were utilized for melanin bleaching with several limitations. For instance, hydrogen peroxide has a weak bleaching ability, and the process is time-consuming (12 h). Meanwhile, potassium permanganate affects the antigenicity of antigens and is unsuitable for immunohistochemical (IHC) staining. In this study, the hypochlorous acid (HClO) solution was applied to hematoxylin-eosin and IHC staining of melanin tissue sections. The study discovered that 1% HClO could completely bleach melanin particles in tumor tissues in a short period (19.95 ± 2.53 min) without compromising the hematoxylin-eosin staining. In addition, 2% HClO was utilized for bleaching at room temperature for 61.17 ± 4.32 minutes after the tissue was incubated with 3,3’-diaminobenzidine in IHC staining. This treatment effectively removed melanin without negatively impacting 3,3’-diaminobenzidine signal expression, thus ensuring that the sections met the necessary diagnostic requirements. Therefore, this method could facilitate pathologists in disease diagnosis of melanin-containing tissues.