title: Effective melanin depigmentation of human and murine ocular tissues: an improved method for paraffin and frozen sections.
abstract: OBJECTIVE
  The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues.
  
  
  METHODS
  Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4) with oxalic acid, and the second 10% hydrogen peroxide (H2O2). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation.
  
  
  RESULTS
  Optimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage.
  
  
  CONCLUSIONS
  Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.
authors.affiliation: Department of Ophthalmology, University Medical Center, Johannes Gutenberg University Mainz, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Manicam
authors.affiliation: Department of Ophthalmology, University Medical Center, Johannes Gutenberg University Mainz, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Pitz
authors.affiliation: Institute of Pathology, University Medical Center, Johannes Gutenberg University Mainz, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Brochhausen
authors.affiliation: Department of Ophthalmology, University Medical Center, Johannes Gutenberg University Mainz, Germany.
authors.forename: Franz H
authors.initials: FH
authors.lastname: Grus
authors.affiliation: Department of Ophthalmology, University Medical Center, Johannes Gutenberg University Mainz, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Pfeiffer
authors.affiliation: Department of Ophthalmology, University Medical Center, Johannes Gutenberg University Mainz, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Gericke
mesh_terms: D000818:Animals; D057886:Bleaching Agents / Q000737:chemistry*; D005123:Eye / Q000737:chemistry*; D005629:Frozen Sections; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D008297:Male; D008543:Melanins / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D019815:Oxalic Acid / Q000737:chemistry; D016612:Paraffin Embedding; D010858:Pigmentation; D011196:Potassium Permanganate / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D057886:Bleaching Agents; D008543:Melanins; D011196:Potassium Permanganate; D019815:Oxalic Acid; D006861:Hydrogen Peroxide