A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue

Lab Invest. 2016 Oct;96(10):1116-27. doi: 10.1038/labinvest.2016.90. Epub 2016 Aug 22.

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.

Publication types

  • Evaluation Study

MeSH terms

  • Formaldehyde
  • Histological Techniques*
  • Hot Temperature*
  • Humans
  • Hydrogen Peroxide*
  • Melanins*
  • Paraffin Embedding

Substances

  • Melanins
  • Formaldehyde
  • Hydrogen Peroxide