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Re‐evaluation of melanin bleaching using warm diluted hydrogen peroxide for histopathological analysis
Author(s) -
Momose Masanobu,
Ota Hiroyoshi,
Hayama Masayoshi
Publication year - 2011
Publication title -
pathology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.73
H-Index - 74
eISSN - 1440-1827
pISSN - 1320-5463
DOI - 10.1111/j.1440-1827.2011.02667.x
Subject(s) - melanin , antigenicity , hydrogen peroxide , chemistry , immunohistochemistry , antigen , pigment , staining , antigen retrieval , phosphate buffered saline , histopathology , microbiology and biotechnology , pathology , biology , chromatography , biochemistry , medicine , immunology , organic chemistry
Excessive amounts of melanin pigments may hamper histopathological assessments of melanocytic lesions by obscuring cellular morphology and hindering antibody–antigen interactions. To determine the optimal melanin‐bleaching conditions for histopathological examination, heavily pigmented melanomas were treated with warm hydrogen peroxide (H 2 O 2 ) diluted with various diluents (1% disodium hydrogen phosphate 12H 2 O ( (Na 2 HPO 4 ); phosphate buffer 0.05 M, pH 7.4 (PB); and PBS 0.05 M, pH 7.4) at varying temperatures (50°C, 55°C, and 60°C) and for varying incubation times (0.5, 1, 2, and 3 h). The effect of the sequential order of antigen retrieval and bleaching on preserving tissue morphology was then evaluated. Additionally, the effect of melanin bleaching using warm diluted H 2 O 2 on the antigenicity of melanoma‐related markers (HMB‐45, MART‐1, and S‐100) and other markers used for histopathology was examined in amelanotic melanomas and tonsil tissue. Optimal and complete bleaching was achieved using warm 3% H 2 O 2 in PB treatment at 55°C for 2 h following antigen retrieval with microwaving or digestion with trypsin. Under these conditions, the tissue morphology and antigenicity of various immunohistochemical markers were also well preserved. Bleaching with warm 3% H 2 O 2 PB is a fast and efficient method of bleaching melanin pigments and performing immunohistochemical examination in heavily melanin‐pigmented lesions.