title: A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue.
abstract: Removal of excessive melanin from heavily pigmented formalin-fixed paraffin-embedded (FFPE) melanoma tissues is essential for histomorphological and molecular diagnostic assessments. Although there have been efforts to address this issue, current methodologies remain complex and time-consuming, and are not suitable for multiple molecular applications. Herein, we have developed a robust and rapid melanin-bleaching methodology for FFPE tissue specimens. Our approach is based on quick bleaching (15 min) at high temperature (80 °C) with 0.5% diluted hydrogen peroxide (H2O2) in Tris-HCl, PBS, or Tris/Tricine/SDS buffer. Immunostaining for Ki-67 and HMB45 was enhanced by bleaching with 0.5% H2O2 in Tris/Tricine/SDS and Tris-HCl, respectively. In addition to histopathological applications, our approach also facilitates recovery of protein and nucleic acid from archival melanin-rich FFPE tissue sections. Protein extracted from bleached FFPE tissues was compatible with western blotting using anti-human GAPDH and AKT antibodies. Our bleaching condition significantly improved RNA quality compared with unbleached tissues without compromising the yield. Notably, the RNA/DNA obtained from bleached tissues was suitable for end point PCR and real-time quantitative RT-PCR. In conclusion, this improved melanin-bleaching method enhances and simplifies immunostaining procedures, and facilitates the use of melanin-rich FFPE tissues for histomorphological and PCR amplification-based molecular assays.
authors.affiliation: Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Joon-Yong
authors.initials: JY
authors.lastname: Chung
authors.affiliation: Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Jiyeon
authors.initials: J
authors.lastname: Choi
authors.affiliation: Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Sears
authors.affiliation: Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Kris
authors.initials: K
authors.lastname: Ylaya
authors.affiliation: Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.|Antibody Characterization Laboratory, Advanced Technology Program, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Candice
authors.initials: C
authors.lastname: Perry
authors.affiliation: Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.|Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
authors.forename: Chel H
authors.initials: CH
authors.lastname: Choi
authors.affiliation: Department of Pathology, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea.
authors.forename: Seung-Mo
authors.initials: SM
authors.lastname: Hong
authors.affiliation: Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.|Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Hanbyoul
authors.initials: H
authors.lastname: Cho
authors.affiliation: Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Brown
authors.affiliation: Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Hewitt
mesh_terms: D005557:Formaldehyde; D006652:Histological Techniques; D006358:Hot Temperature; D006801:Humans; D006861:Hydrogen Peroxide; D008543:Melanins; D016612:Paraffin Embedding
chemical_list: D008543:Melanins; D005557:Formaldehyde; D006861:Hydrogen Peroxide