Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study

Abstract

In fluorescence microscopy, light radiation can be used to bleach fluorescent molecules in formalin-fixed and paraffin-embedded (FFPE) samples, in order to increase the ratio between signal of interest and background autofluorescence. We tested if the same principle can be exploited in bright field microscopy to bleach pigmented melanoma FFPE sections together with cell morphology maintenance. After dewaxing and rehydration, serial FFPE sections of a feline diffuse iris melanoma, a canine dermal melanoma, a gray horse dermal melanoma and a swine cutaneous melanoma were irradiated with visible light for 1, 2, 3, 4 and 5 days, prior to Hematoxylin & Eosin staining. Complete bleaching was obtained after 1-day treatment in feline and swine melanomas, while 2 and 3 days were required in canine and equine neoplasms, respectively. In all treated samples, cell morphology was maintained. Photo-induced bleaching combined with immunohistochemistry was tested after a 3-day photo-treatment using five different markers. According to the literature, in all samples neoplastic cells stained positive for vimentin, S100 and PNL2, while negative for FVIII and pancytokeratin. In conclusion, visible light can be effectively exploited to bleach pigmented melanoma FFPE sections prior to perform routine histochemical and immunohistochemical stains.

Light sheet fluorescence microscopy of cleared human eyes

“…The human eye has a high concentration of melanin and requires specific processes for bleaching. To overcome this, we used a combination of photo-bleaching, a technique used in histopathological protocols for highly pigmented samples of melanoma 22 , and oxygen peroxide (H2O2) 23 , most recently used in clearing of the mouse eye 16 . Next, to improve penetration of antibodies through the vitreous and sclera, we used an immunolabeling protocol based on the modulation of antibody-antigen binding 24 , 25 .…”

Characterization of Spontaneous Melanization by Fluorescence Spectroscopy: A Basis for Analytical Application to Biological Substrates

“…In histopathology, melanin can hinder the bright-field-microscope observation of morphology and expression markers of stained and immune-stained cells and tissues. This problem can be solved with a suitable light irradiation of the tissue slice to induce photobleaching and the consequent disappearance of the dark pigment, as verified for melanoma samples from different veterinary sources [ 41 ]. Conversely, sophisticated techniques such as multiphoton excitation and lifetime spectroscopy and imaging focus on melanin as an optical parameter for the real-time, in vivo screening of skin disease and diagnosis of melanoma [ 42 , 43 , 44 , 45 , 46 , 47 ].…”