Abstract
PurposeThe 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.MethodsParaffin-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.ResultsOptimal 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.ConclusionsWarm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.
Highlights
The eye is a specialized organ that comprises several layers of melanin-pigmented tissues with differing embryonic origins
Melanocytes in the ocular tissue are localized in the uvea, retinal pigment epithelium and surface epithelia, with heavy pigmentation concentrated in the uvea [1]
This is especially important in the diagnosis and prognosis of melanomas, in which heavily pigmented tissues mask the interpretation of malignancy in histological sections [6]
Summary
The eye is a specialized organ that comprises several layers of melanin-pigmented tissues with differing embryonic origins. Melanin is a naturally occurring complex oligometric material that consists of various monomers, which are the oxidation and tautomerization products of DOPA (dihydroxyphenylalanine) derived from the amino acid tyrosine [3,4] This pigment is characteristically brown-black (eumelanin) and the difference in melanization found in various regions of the eye has been attributed to the specific functional relevance of melanin [5]. Bleaching techniques are important to remove excessive melanin granules that obscure the morphology of tissues subsequently interfering with differential diagnosis This is especially important in the diagnosis and prognosis of melanomas, in which heavily pigmented tissues mask the interpretation of malignancy in histological sections [6]. Melanoma is the most common primary malignancy of the eye [7], and the use of melanin bleaching is indispensable in the interpretation of richly pigmented melanocytic lesions
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