To select the optimal immunohistochemical staining method for pigmented melanoma
Abstract Melanin granules may be difficult to distinguish from the brown reaction product of 3,3’-diaminobenzidine (DAB) in immunostaining. To identify a reliable immunostaining protocol for pigmented melanoma that preserves tissue integrity while enabling clear observation of antigen-antibody bindi...
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Nature Portfolio
2025-08-01
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| Online Access: | https://doi.org/10.1038/s41598-025-13085-1 |
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| author | Juan Lin Zhijie You Xin Chen |
| author_facet | Juan Lin Zhijie You Xin Chen |
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| description | Abstract Melanin granules may be difficult to distinguish from the brown reaction product of 3,3’-diaminobenzidine (DAB) in immunostaining. To identify a reliable immunostaining protocol for pigmented melanoma that preserves tissue integrity while enabling clear observation of antigen-antibody binding. Ki67 and the human melanoma black 45 (HMB45) immunostaining was performed on 30 pigmented melanoma specimens using three methods: (1) Unbleached control, (2) Bleached with 5% H₂O₂, (3) Alkaline Phosphatase–3-Amino-9-Ethylcarbazole (AP-AEC). Compared with the unbleached control, the coincidence rates of Ki67 and HMB45 expression in bleached with 5% H2O2 group were 90.9% and 86.4%, respectively, and the one in AP-AEC group were 81.8% and 90.9%, respectively. No significant differences (all p > 0.05) in Ki67 expression and HMB45 expression parameters (percentage, staining intensity and H-score) between bleached group vs. unbleached control and AP-AEC group vs. control across all comparative measured parameters. A 25% (4/16) tissue damage rate occurred in heavily pigmented specimens subjected to the bleaching group, and non-specific staining were observed in AP-AEC processed samples. For lightly pigmented melanoma, bleached with 5% H2O2 method optimally balanced tissue preservation and staining reliability. Non-specific immunostaining was observed in the AP-AEC group. For heavily pigmented melanoma, AP-AEC method minimized tissue damage despite minor non-specificity. |
| format | Article |
| id | doaj-art-b33d972e555f4546b16065c7d7e4e168 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-08-01 |
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| spelling | doaj-art-b33d972e555f4546b16065c7d7e4e1682025-08-24T11:29:20ZengNature PortfolioScientific Reports2045-23222025-08-011511810.1038/s41598-025-13085-1To select the optimal immunohistochemical staining method for pigmented melanomaJuan Lin0Zhijie You1Xin Chen2Shengli Clinical Medical College, Fujian Medical UniversityShengli Clinical Medical College, Fujian Medical UniversityShengli Clinical Medical College, Fujian Medical UniversityAbstract Melanin granules may be difficult to distinguish from the brown reaction product of 3,3’-diaminobenzidine (DAB) in immunostaining. To identify a reliable immunostaining protocol for pigmented melanoma that preserves tissue integrity while enabling clear observation of antigen-antibody binding. Ki67 and the human melanoma black 45 (HMB45) immunostaining was performed on 30 pigmented melanoma specimens using three methods: (1) Unbleached control, (2) Bleached with 5% H₂O₂, (3) Alkaline Phosphatase–3-Amino-9-Ethylcarbazole (AP-AEC). Compared with the unbleached control, the coincidence rates of Ki67 and HMB45 expression in bleached with 5% H2O2 group were 90.9% and 86.4%, respectively, and the one in AP-AEC group were 81.8% and 90.9%, respectively. No significant differences (all p > 0.05) in Ki67 expression and HMB45 expression parameters (percentage, staining intensity and H-score) between bleached group vs. unbleached control and AP-AEC group vs. control across all comparative measured parameters. A 25% (4/16) tissue damage rate occurred in heavily pigmented specimens subjected to the bleaching group, and non-specific staining were observed in AP-AEC processed samples. For lightly pigmented melanoma, bleached with 5% H2O2 method optimally balanced tissue preservation and staining reliability. Non-specific immunostaining was observed in the AP-AEC group. For heavily pigmented melanoma, AP-AEC method minimized tissue damage despite minor non-specificity.https://doi.org/10.1038/s41598-025-13085-1Melanin bleachingImmunostainingPigmented melanomaAECDABH2O2 |
| spellingShingle | Juan Lin Zhijie You Xin Chen To select the optimal immunohistochemical staining method for pigmented melanoma Scientific Reports Melanin bleaching Immunostaining Pigmented melanoma AEC DAB H2O2 |
| title | To select the optimal immunohistochemical staining method for pigmented melanoma |
| title_full | To select the optimal immunohistochemical staining method for pigmented melanoma |
| title_fullStr | To select the optimal immunohistochemical staining method for pigmented melanoma |
| title_full_unstemmed | To select the optimal immunohistochemical staining method for pigmented melanoma |
| title_short | To select the optimal immunohistochemical staining method for pigmented melanoma |
| title_sort | to select the optimal immunohistochemical staining method for pigmented melanoma |
| topic | Melanin bleaching Immunostaining Pigmented melanoma AEC DAB H2O2 |
| url | https://doi.org/10.1038/s41598-025-13085-1 |
| work_keys_str_mv | AT juanlin toselecttheoptimalimmunohistochemicalstainingmethodforpigmentedmelanoma AT zhijieyou toselecttheoptimalimmunohistochemicalstainingmethodforpigmentedmelanoma AT xinchen toselecttheoptimalimmunohistochemicalstainingmethodforpigmentedmelanoma |