Effective melanin depigmentation of human and murine ocular tissues : an improved method for paraffin and frozen sections

Abstract

PURPOSE: 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 degrees 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.