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Home / Equine Research Bank / Eur J Histochem / Bleaching melanin in formalin-fixed and paraffin-embedded me...
European journal of histochemistry : EJH2019; 63(4); doi: 10.4081/ejh.2019.3071

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 and 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.
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Publication Date: 2019-10-31 PubMed ID: 31833329PubMed Central: PMC6829521DOI: 10.4081/ejh.2019.3071Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study explores the use of visible light to bleach melanin in formalin-fixed and paraffin-embedded (FFPE) melanoma samples for better microscopy results. The researchers established that sufficient light exposure, with durations varying between species, effectively bleached melanin, while preserving cell structure and compatibility with immunohistochemistry.

Research Methodology

  • The researchers used samples including a feline diffuse iris melanoma, a canine dermal melanoma, a gray horse dermal melanoma, and a swine cutaneous melanoma. These samples were formalin-fixed and paraffin-embedded (FFPE).
  • Following the process of dewaxing and rehydration, these FFPE sections were exposed to visible light for periods ranging from 1 to 5 days. This was done prior to staining them with Hematoxylin and Eosin

Observations and Findings

  • After 1-day light treatment, complete bleaching was achieved in the feline and swine melanomas.
  • For the canine and equine melanomas, bleaching required 2 to 3 days respectively.
  • In all cases, the cellular morphology was preserved which is crucial for maintaining the integrity of the samples for further analysis.
  • Besides, the microscopic analysis could be performed using bright field microscopy as well.

Immunohistochemistry Post Bleaching

  • Following a 3-day light exposure, the researchers performed immunohistochemistry using five different markers.
  • The neoplastic cells in all samples were found to test positive for vimentin, S100, and PNL2, and negative for FVIII and pancytokeratin, according to existing literature.

Conclusion

  • Overall, the study validates the use of visible light to effectively bleach pigmented melanoma FFPE sections.
  • This method not only preserves the cell morphology but also facilitates further histochemical and immunohistochemical stains, opening up opportunities for better visualization and diagnostic potential for melanomas.

Cite This Article

APA
Pigoli C, Gibelli LR, Caniatti M, Moretti L, Sironi G, Giudice C. (2019). Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study. Eur J Histochem, 63(4). https://doi.org/10.4081/ejh.2019.3071

Publication

European journal of histochemistry : EJH
ISSN: 2038-8306
NlmUniqueID: 9207930
Country: Italy
Language: English
Volume: 63
Issue: 4

Researcher Affiliations

Pigoli, Claudio
  • Department of Veterinary Medicine (DIMEVET), University of Milan, Lodi. claudio.pigoli@unimi.it.
Gibelli, Lucia Rita
    Caniatti, Mario
      Moretti, Luca
        Sironi, Giuseppe
          Giudice, Chiara

            MeSH Terms

            • Animals
            • Antibodies, Monoclonal, Murine-Derived / immunology
            • Cats
            • Dogs
            • Formaldehyde / chemistry
            • Goats
            • Horses
            • Immunohistochemistry
            • Light
            • Melanins / radiation effects
            • Melanoma / pathology
            • Melanoma / veterinary
            • Mice
            • Paraffin Embedding
            • Photobleaching
            • Pilot Projects
            • Rabbits
            • S100 Proteins / immunology
            • Swine
            • Temperature
            • Vimentin / immunology

            Conflict of Interest Statement

            Conflict of interest: the authors declare they have no competing interests.

            References

            This article includes 30 references
            1. Solano F. Melanins: Skin pigments and much more—Types, structural models, biological functions, and formation routes.. New J Sci 2014;2014.
              doi: 10.1155/2014/498276google scholar: lookup
            2. Bush WD, Garguilo J, Zucca FA, Albertini A, Zecca L, Edwards GS. The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface.. Proc Natl Acad Sci US 2006;103:14785-9.
              pmc: PMC1595429pubmed: 17001010
            3. Ito S, Wakamatsu K, Sarna T. Photodegradation of eumelanin and pheomelanin and its pathophysiological implications.. Photochem Photobiol 2018;94: 409-20.
              pubmed: 28873228doi: 10.1111/php.12837google scholar: lookup
            4. Szewczyk G, Zadlo A, Sarna M, Ito S, Wakamatsu K, Sarna T. Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion.. Pigment Cell Melanoma Res 2016;29:669-78.
              pubmed: 27505632doi: 10.1111/pcmr.12514google scholar: lookup
            5. Neumann M, Gabel D. Simple method for reduction of autofluorescence in fluorescence microscopy.. J Histochem Cytochem 2002;50:437-9.
              pubmed: 11850446doi: 10.1177/002215540205000315google scholar: lookup
            6. Duong H, Han M. A multispectral LED array for the reduction of background autofluorescence in brain tissue.. J Neurosci Methods 2013;220:46-54.
              pmc: PMC3856220pubmed: 23994358doi: 10.1016/j.jneumeth.2013.08.018google scholar: lookup
            7. Kumar BS, Sandhyamani S, Nazeer SS, Jayasree RS. Rapid and simple method of photobleaching to reduce background autofluorescence in lung tissue sections.. Indian J Biochem Biophys 2015;52:107-10.
              pubmed: 26040118
            8. Sun Y, Ip P, Chakrabartty A. Simple elimination of background fluorescence in formalin- fixed human brain tissue for immunofluorescence microscopy.. J Vis Exp 2017;:e56188.
              pmc: PMC5614400pubmed: 28892031doi: 10.3791/56188google scholar: lookup
            9. Schmid FX. Biological macromolecules: UV-visible spectrophotometry.. Encyclopedia of Life Sciences Robinson S., Ayres E., Editors; J. Wiley & Sons, Hoboken; 2001.
              doi: 10.1038/npg.els.0003142google scholar: lookup
            10. Jordan T, Williams D, Criswell S, Wang Y. Comparison of bleaching protocols utilizing hematoxylin and eosin stain and immunohistochemical proliferation marker MCM3 in pigmented melanomas.. J Histotechnol 2019;16:1-6.
              pubmed: 31416403doi: 10.1080/01478885.2019.1649886google scholar: lookup
            11. Elvidge CD, Keith DM, Tuttle BT, Baugh KE. Spectral identification of lighting type and character.. Sensors 2010;10:3961-88.
              pmc: PMC3274255pubmed: 22319336doi: 10.3390/s100403961google scholar: lookup
            12. Wiggans KT, Reilly CM, Kass PH, Maggs DJ. Histologic and immunohistochemical predictors of clinical behavior for feline diffuse iris melanoma.. Vet Ophthalmol 2016;19(S1):44-55.
              pubmed: 26805705doi: 10.1111/vop.12344google scholar: lookup
            13. Nishiya AT, Massoco CO, Felizzola CR, Perlmann E, Batschinski K, Tedardi MV. Comparative aspects of canine melanoma.. Vet Sci 2016;3pii:E7.
              pmc: PMC5644618pubmed: 29056717doi: 10.3390/vetsci3010007google scholar: lookup
            14. Teixeira RB, Rendahl AK, Anderson SM, Mickelson JR, Sigler D, Buchanan BR. Coat color genotypes and risk and severity of melanoma in gray quarter horses.. J Vet Intern Med 2013;27:1201-8.
              pubmed: 23875712doi: 10.1111/jvim.12133google scholar: lookup
            15. Grossi AB, Hyttel P, Jensen HE, Leifsson PS. Porcine melanotic cutaneous lesions and lymph nodes: immunohistochemical differentiation of melanocytes and melanophages.. Vet Pathol 2015;52:83-91.
              pubmed: 24503437doi: 10.1177/0300985814521637google scholar: lookup
            16. Swope VB, Abdel-Malek ZA. MC1R: Front and center in the bright side of dark eumelanin and DNA repair.. Int J Mol Sci 2018;19 pii: E2667.
              pmc: PMC6163888pubmed: 30205559doi: 10.3390/ijms19092667google scholar: lookup
            17. Grahn BH, Peiffer RL, Cullen CL, Haines DM. Classification of feline intraocular neoplasms based on morphology, histochemical staining, and immunohistochemical labelling.. Vet Ophthalmol 2006;9:395-403.
              pubmed: 17076872doi: 10.1111/j.1463-5224.2006.00479.xgoogle scholar: lookup
            18. Smedley RC, Lamoureux J, Sledge DG, Kiupel M. Immunohistochemical diagnosis of canine oral amelanotic melanocytic neoplasms.. Vet Pathol 2011;48:32-40.
              pubmed: 21078882doi: 10.1177/0300985810387447google scholar: lookup
            19. Nordio L, Fattori S, Vascellari M, Giudice C. Evidence of vasculogenic mimicry in a palpebral melanocytoma in a dog.. J Comp Pathol 2018;162:43-46.
              pubmed: 30060841doi: 10.1016/j.jcpa.2018.06.003google scholar: lookup
            20. Ramos-Vara JA, Frank CB, DuSold D, Miller MA. Immunohistochemical expression of melanocytic antigen PNL2, Melan A, S100, and PGP 9.5 in equine melanocytic neoplasms.. Vet Pathol 2014;51:161-6.
              pubmed: 23370093doi: 10.1177/0300985812471545google scholar: lookup
            21. Bottegaro NB, Gotic J, Babic NP, Zagradisnik LM, Kurilj AG. Abrupt exacerbation of a mid-tail melanoma during late pregnancy in a bay Holsteiner mare.. Wien Tierärzt Monat 2018;105:25-31.
            22. Perez J, García PM, Bautista MJ, Millán Y, Ordás J, Martín de las Mulas J. Immunohistochemical characterization of tumor cells and inflammatory infiltrate associated with cutaneous melanocytic tumors of Duroc and Iberian swine.. Vet Pathol 2002;39:445-51.
              pubmed: 12126147doi: 10.1354/vp.39-4-445google scholar: lookup
            23. Shen H, Wu W. Study of melanin bleaching after immunohistochemistry of melanin-containing tissues.. Appl Immunohistochem Mol Morphol 2015;23:303-7.
              pmc: PMC4424098pubmed: 24710084doi: 10.1097/pai.0000000000000075google scholar: lookup
            24. Foss AJ, Alexander RA, Jefferies LW, Lightman S. Immunohistochemical techniques: the effect of melanin bleaching.. Br J Biomed Sci 1995;52:22-5.
              pubmed: 7549602
            25. Orchard GE, Calonje E. The effect of melanin bleaching on immunohistochemical staining in heavily pigmented melanocytic neoplasms.. Am J Dermatopathol 1998;20:357-61.
              pubmed: 9700373doi: 10.1097/00000372-199808000-00006google scholar: lookup
            26. Driessens N, Versteyhe S, Ghaddhab C, Burniat A, De Deken X, Van Sande J. Hydrogen peroxide induces DNA single- and double-strand breaks in thyroid cells and is therefore a potential mutagen for this organ.. Endocr Relat Cancer 2009;16:845-56.
              pubmed: 19509065doi: 10.1677/erc-09-0020google scholar: lookup
            27. Olson MJ. DNA strand breaks induced by hydrogen peroxide in isolated rat hepatocytes.. J Toxicol Environ Health 1988;23:407-23.
              pubmed: 3351984doi: 10.1080/15287398809531123google scholar: lookup
            28. McDonald RJ, Pan LC, St George JA, Hyde DM, Ducore JM. Hydrogen peroxide induces DNA single strand breaks in respiratory epithelial cells.. Inflammation 1993;17:715-22.
              pubmed: 8112830doi: 10.1007/bf00920476google scholar: lookup
            29. An S, Lee E, Choe E. Effects of solubility characteristics of sensitiser and pH on the photooxidation of oil in tuna oiladded acidic O/W emulsions.. Food Chem 2011;128:358-63.
              pubmed: 25212142doi: 10.1016/j.foodchem.2011.03.034google scholar: lookup
            30. Bancirova M. Sodium azide as a specific quencher of singlet oxygen during chemiluminescent detection by luminol and Cypridina luciferin analogues.. Luminescence 2011;26:685-8.
              pubmed: 21491580doi: 10.1002/bio.1296google scholar: lookup
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