Bleaching Melanin in Formalin- Fixed and Paraffin-Embedded Melanoma

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Bleaching melanin in formalin- different and partly unknown molecular fixed and paraffin-embedded features. In general, melanins can be Correspondence: Claudio Pigoli, Department defined as a heterogeneous group of mole- of Veterinary Medicine, University of Milan, melanoma specimens using cules deriving from the oxidation of pheno- Via dell'Università 6, 26900 Lodi, Italy. visible light: A pilot study lic compounds with polymerization of the Tel. +39.02.50334165. resulting chemical products. In animals, E-mail: [email protected] 1 2 melanins are subdivided into eumelanins, Claudio Pigoli, Lucia Rita Gibelli, Key words: Bright-field microscopy; canine; 1 3 black to brown in color, and pheomelanins, Mario Caniatti, Luca Moretti, 1 equine; feline; light radiation; photobleaching; 1 1 reddish to yellowish. A third subtype of swine. Giuseppe Sironi, Chiara Giudice melanin, called neuromelanin and often 1Department of Veterinary Medicine, associated to the afore-mentioned groups, is Contributions: CP, LRG, MC, LM, GS, CG, University of Milan, Lodi composed of a mixture of eumelanins and study design, data analysis and interpretation; 2Istituto Zooprofilattico Sperimentale pheomelanins. In particular, it has been LM, performing of the light source spectral measurement; CP, performing of the photo- della Lombardia e dell’Emilia Romagna, hypothesized that neuromelanin granules consist of a pheomelanin core surrounded bleaching protocol, histochemical and Milan 2 immunohistochemical stains; CP, LRG, MC, 3 by a eumelanin layer. Department of Physics, Politecnico di GS, CG, viewed the histological specimens. In animals, melanin granules are pres- Milano, Milan, Italy All the authors have drafted the manuscript ent in various sites including integumentary and reviewed the work critically, have read system, eye, central nervous system and and approved the final manuscript and agreed inner ear. Given its presence in a wide range to be accountable for all aspects of the work. of tissues, it is not surprising that melanin Abstract performs different tasks ranging from mere- Conflict of interest: the authors declare they In fluorescence microscopy, light radia- ly mechanical ones to those of radical scav- have no competing interests. tion can be used to bleach fluorescent mole- engers. The main role of melanin is to pro- Acknowledgements: We would like to thank cules in formalin-fixed and paraffin-embed- tect living organisms from the harmful Massimo Datteri and Laura Tassielli from the ded (FFPE) samples, in order to increase the effects of electromagnetic radiations. In Istituto Zooprofilattico Sperimentale della ratio between signal of interest and back- particular, it is able to absorb radiations in a Lombardia e dell’Emilia Romagna for the ground autofluorescence. We tested if the wide range of wavelengths from the ultravi- technical support, cutting and staining of his- same principle can be exploited in bright olet to the visible spectrum. The energy tological sections. We would also like to thank field microscopy to bleach pigmented absorbed is then redistributed as heat.1 Stefano Tomasoni from DI.C.S.EL srl for his melanoma FFPE sections together with cell Energy absorption is also followed by redox valuable advices regarding the world of LED morphology maintenance. After dewaxing reactions that ultimately can lead to forma- light sources. and rehydration, serial FFPE sections of a tion of molecules having spectral absorp- Received for publication: 9 September 2019. feline diffuse iris melanoma, a canine dermal tion properties that differ from the original Accepted for publication: 15 October 2019. melanoma, a gray horse dermal melanoma polymer. Since melanins absorb both visible and a swine cutaneous melanoma were irra- and not visible light radiation, their oxida- This work is licensed under a Creative diated with visible light for 1, 2, 3, 4 and 5 tion can be achieved both by high-energy Commons Attribution-NonCommercial 4.0 days, prior to Hematoxylin & Eosin staining. radiation, such as ultraviolet rays, and by International License (CC BY-NC 4.0). Complete bleaching was obtained after 1-day low-energy radiation, such as visible light. treatment in feline and swine melanomas, The phenomenon by which a molecule ©Copyright: the Author(s), 2019 while 2 and 3 days were required in canine undergoes oxidation by means of light irra- Licensee PAGEPress, Italy and equine neoplasms, respectively. In all 3 European Journal of Histochemistry 2019; 63:3071 diation is called photo-oxidation. doi:10.4081/ejh.2019.3071 treated samples, cell morphology was main- Although the exact mechanism of melanin tained. Photo-induced bleaching combined photo-oxidation has not yet been explained with immunohistochemistry was tested after in all its aspects, several studies have shown a 3-day photo-treatment using five different that during ultraviolet and visible light irra- Materials and Methods markers. According to the literature, in all diation, singlet oxygen and superoxide samples neoplastic cells stained positive for anions are produced and rapidly quenched Samples vimentin, S100 and PNL2, while negative by melanin. The oxidation induced by these for FVIII and pancytokeratin. In conclusion, reactive oxygen species is considered to In this study, four FFPE samples of pig- visible light can be effectively exploited to play an important role in the photo-degrada- mented melanocytic neoplasms were used. bleach pigmented melanoma FFPE sections tion of melanin itself.3,4 Visible light is rou- In particular, a grey horse dermal melanoma prior to perform routine histochemical and tinely used, in our laboratory, to damage the and a swine cutaneous melanoma were immunohistochemical stains. molecules responsible for background auto- retrieved from the archives of the Istituto fluorescence, thus making them no longer Zooprofilattico Sperimentale della fluorescent, prior to perform immunofluo- Lombardia e dell’Emilia Romagna rescence assays on formalin-fixed and (IZSLER), while a case of feline diffuse iris paraffin-embedded (FFPE) samples.5-8 In melanoma and a case of canine dermal Introduction the present study, we investigated if the melanoma were obtained from the archives Melanins is a generic term derived from same principle could also be exploited in of the Department of Veterinary Medicine, the Greek word “melanos” meaning dark. bright field microscopy to bleach FFPE University of Milan. For each sample, six 4 Although the term melanin is often used to samples of pigmented melanoma, prior to µm-thick serial sections were cut and identify a single entity, it actually encom- perform routine histochemical and placed on SuperFrost Plus™ adhesion passes a number of pigments widely distrib- immunohistochemical stains, ensuring the slides (Thermo Scientific, Waltham, MA, uted in living beings and characterized by maintenance of cell morphology. USA).

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Light source and emission spectrum buffer maintained at 24°C (the temperature different markers were tested: three of them evaluation reached by the sections during photo-treat- (vimentin, S100 and PNL2) are commonly In this study a 300 W ViugreumTM spot- ment) and in the dark inside a thermostat used in the melanocytic neoplasm histolog- light (Guangdong Zhongshan Yilangde incubator (Cavallo srl, Buccinasco, MI, ical identification workflow, while two Illumination CO. Ltd., Zhongshan City, Italy). Every 24 h, for 5 days, a section for (pancytokeratin and FVIII) are usually not China) containing a 594-phosphor white each sample was removed from the Petri expressed by melanoma cells. Sections LED panel with an emission divergence of dishes and stored with the control sections. were dewaxed in xylene and rehydrated 120° and emitting a 36000 lum and 6500 K At the end of the fifth day, to minimize the through graded alcohols; endogenous per- light, was used. Since the manufacturer of possible differences given by a non-con- oxidase activity was blocked with 0.3% the spotlight supplies only the color temper- temporary histochemical staining, all histo- H2O2 in methanol for 30 min. Sections were ature of the light emitted but not the specific logical sections were jointly stained with then dipped in Petri dishes containing emission spectrum, the latter was measured hematoxylin-eosin (H&E). 0.05% sodium azide TRIS buffer and pho- using a HR2000+ spectrometer (Ocean tobleached for 3 days. The duration of the Photobleaching protocol combined photo-treatment was chosen after the evalu- Optics, Largo, Florida), which has a wave- with immunohistochemical stain length range of detection from 190 nm to ation of the results of photobleaching and In the immunohistochemical assay, five 1100 nm. The light of the lamp has been H&E combined protocol: the treatment able coupled to the spectrometer through an optical fiber. Photobleaching apparatus The photobleaching apparatus has been made similarly to the one described by Duong and Ha.6 The spotlight was placed in a room maintained at 4°C and placed so that the LED panel was facing upwards and that 4.5 cm thick reflective spacers could be placed on the spotlight protective glass. With this configuration the distance between the samples and the LED panel was 8.8 cm. Above the spacers, two 12-cm square Petri dishes were placed, filled with TRIS buffer pH 7.6 added with 0.05% sodium azide, in order to inhibit bacterial growth (Figure 1). A glass mirror was posi- tioned on the Petri dishes so that the light not directly absorbed by the samples could, at least in part, be redirected towards them. Figure 1. Photobleaching apparatus. The spotlight was placed so that the LED panel was fac- With this configuration of the apparatus, the ing upwards and that reflective spacers could be placed on the spotlight protective glass. Above amount of light impinging directly on the the spacers, two 12-cm square Petri dishes, filled with TRIS buffer added with 0.05% sodium samples from the LEDs has been calculated azide, were placed. In the image, the covering glass mirror is not visible. to be around 0.455 mW/cm2 (derived from the LED panel parameters declared by the manufacturer and measured spectrum). Temperatures reached by the sections during photobleaching treatment To evaluate the temperature reached by the sections during treatment, during a preliminary experiment, an alcohol thermome- ter was placed in the TRIS buffer-filled Petri dishes of the photobleaching apparatus. The temperatures were recorded before starting the treatment and, once started, at 1-h intervals for 12 h. Photobleaching protocol combined with routine histochemical stain Sections were dewaxed in xylene, rehydrated through graded alcohols and then placed in 0.05% sodium azide TRIS buffer. The sections were inserted into the Petri Figure 2. Light source emission spectrum. Emitted radiation is in the visible range (~400- 750 nm), showing main peaks in the blue and green-yellow ranges. The colored stripe in dishes and the photobleaching protocol was the bottom of the graph roughly represent how different wavelengths would be perceived started. A control section for each sample by human eye. was dipped in 0.05% sodium azide TRIS

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Figure 3. Effect of the photo-treatment on feline diffuse iris melanoma (A), canine dermal melanoma (B), gray horse dermal melanoma (C) and swine cutaneous melanoma (D) FFPE samples. In the figure untreated (0) and 1-day (1), 2-day (2), 3-day (3), 4-day (4), 5-day (5) treated sections are shown. A0-5) In the feline sample, after a 24-h photobleaching treatment, neoplastic cells were completely depigmented and no differences were noted for longer treatments; in all treated sections, cell morphology was well maintained. B0-5) In the canine sample, a 24-h treatment resulted in a considerable melanin degradation; still, single or aggregated neoplastic melanocytes, either totally black or characterized by a black-brownish cytoplasm, were observed; following a 2-days treatment, neoplastic cells were entirely depigmented and no further changes were noted in the 3-, 4- and 5-days treatments. In all treated sections, cell morphology was well maintained. C0-5) In the equine sample after a 24-h treatment, a considerable part of the section was still pigmented, with melanocytes characterized by a totally black cytoplasm or variable numbers of intracytoplasmic round black granules; after a 2-days treatment, occasional melanocytes with intracytoplasmic granules were still present; melanin was not observed in the 3-, 4- and 5-days treated sections. In all treated sections, cell morphology was well maintained. D0-5) In the swine sample, pigmented cells were completely bleached after a 24-h treatment; no changes were observed following longer treatments; in all treated sections, cell morphology was well maintained. H&E staining.

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to ensure the complete depigmentation of source (Figure 2) used to perform the treat- neoplastic cells, and iris, ciliary and retinal all samples was applied. Following photo- ments, was found to be distributed in the pigment epithelial cells were completely bleaching, different antigen retrieval meth- visible range of light radiation (~400–750 bleached and no differences were noted for ods were applied: pepsin enzymatic diges- nm), with main peaks in the blue and green- longer treatments (Figure 4). In all treated tion (Digest-All Invitrogen; Thermo Fisher yellow bands, around 460 nm and 540 nm sections, morphology of neoplastic and Scientific, Carlsbad, CA, USA) for 15 min respectively. In particular, it is important to non-neoplastic cells was maintained. at 37°C was used for pancytokeratin and remark that no emission was present in the Canine dermal melanoma (Figure 3B). FVIII; heat-induced antigen retrieval with UV wavelength range. A 24-h treatment resulted in a considerable microwave oven for 10 min at 567 W in cit- melanin degradation. Still, single or aggre- rate buffer pH 6.0 for vimentin; heat- Temperatures reached by the sec- gated neoplastic melanocytes, either totally induced antigen retrieval with microwave tions during photobleaching treat- black or characterized by a black-brownish oven for 10 min at 567 W in EDTA buffer ment cytoplasm, were observed. Following a 2- pH 8.5 for PNL2. No antigen retrieval was The temperatures recorded inside the days treatment, neoplastic cells were entire- performed for S100. Blocking was obtained Petri dishes underwent a rapid increase, ris- ly depigmented and no further changes with a 1-h incubation at room temperature ing from 4°C before starting the treatment were noted in the 3-, 4- and 5-days treat- (RT) with horse normal serum for mono- to 24°C after an hour from the beginning of ments. In all treated sections, morphology clonal and goat normal serum for polyclon- the latter, remaining then stable at the same of neoplastic and non-neoplastic cells was al antibodies. Mouse monoclonal anti-pan- temperature for the following 11 h. maintained. Furthermore, the presence of an cytokeratin (clones AE1/AE3, Dako, amelanotic area within the neoplasms Carpinteria, CA, USA; 1:2000 dilution), Melanin photobleaching and cell allowed to observe that the morphology of morphology maintenance anti-vimentin (Clone 3B4, Dako; 1:1000 amelanotic neoplastic cells did not change dilution) and anti-PNL2 (Clone PNL2, Feline diffuse iris melanoma (Figure following treatments of different duration Monosan, Uden, The Netherlands; 1:25 3A). After a 24-h photobleaching treatment, (Figure 5). dilution), and rabbit polyclonal anti-FVIII (Dako; 1:200 dilution) and anti-S100 (Dako; 1:500 dilution) antibodies were used. In negative controls, primary antibodies were replaced with normal rabbit or mouse serum (non-immune serum; Dako). Primary antibodies were incubated overnight at 4°C. Immunohistochemical reaction was visualized through an avidin- biotin complex method using biotinylated goat anti-rabbit (Vector Laboratories, Burlingame, CA, USA) and horse anti- mouse (Vector Laboratories) antibodies, and AEC (3-amino-9-ethylcarbazole) sub- strate-chromogen kit (Vector Laboratories). Sections were counterstain with Mayer’s hematoxylin. Figure 4. Ciliary bodies, pars plicata. Comparison of same field in a not-photobleached (A) and a 1-day photo-treated (B) section. Pigmented epithelium in specimen B was com- Evaluation of melanin photo-degra- pletely bleached and cell morphology was excellently maintained. H&E staining. dation, cell morphology maintenance and immunohistochemical results To assess pigment photo-degradation and maintenance of cell morphology, sample sections subjected to different treatment duration have been compared each other and with control sections. Cell morphology was assessed on both neoplastic and all other cellular types present in sections (e.g., vessel endothelium, stromal cells, epithelial cell). For the evaluation of the immunohistochemical assay, the obtained results (positivity or negativity of the neoplastic cells for different markers) were compared with the expected results based on available literature.

Results Figure 5. Canine dermal melanoma. Comparison of a not- (A) and a 5-day (B) photo- treated section showing the border between an amelanotic and a melanotic area. Light source emission spectrum Morphology of neoplastic cells is excellently maintained even after the treatment of max- imum duration. H&E staining. The emission spectrum of the light

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Gray horse dermal melanoma (Figure ing fluorophores more seriously affected by days were required to obtain the same result 3C). After a 24-h treatment, a considerable irradiation of chosen wavelengths, com- in the canine and equine samples, respec- part of the section was still pigmented, with pared to those that are not able to adsorb the tively. The long exposure time required by melanocytes characterized by a totally same spectral range.5,6 In the present work, the equine sample to reach complete depig- black cytoplasm or variable numbers of following the same principle, we evaluate mentation can be explained in light of coat intracytoplasmic round black granules. whether it is possible to exploit melanin color and melanoma pathogenesis in gray After a 2-days treatment, occasional absorption spectra to determine its degrada- horses. In these horses, dermal melanoma melanocytes with intracytoplasmic granules tion in FFPE sections. To obtain a more tar- development is linked to specific duplica- were still present. Melanin was not geted effect, we preferred to use an only- tions of genomic regions containing binding observed in the 3-, 4- and 5-days treated visible-light emitting light source to avoid sites for proteins involved in melanocyte sections. In all treated sections, morphology an excessively deleterious effect on the gene expression and metabolism. This of neoplastic and non-neoplastic cells was samples deriving from the use of ultraviolet genomic modification brings to eumelanin maintained. radiations. The latter, characterized by a overproduction and to an increased prolifer- Swine cutaneous melanoma (Figure higher energy content compared to visible ative boost.14 It can be therefore hypothe- 3D). In swine cutaneous pigmented radiations, are indeed absorbed also by sized that the predominant pigment in the melanoma, pigmented cells were complete- nucleic acids, peptide groups and aromatic equine sample was eumelanin, less prone to ly bleached after a 24-h treatment. No residues of proteins, most likely affecting photo-degradation than pheomelanin.3,16 changes were observed following longer the specificity of immune-histochemical Moreover, since the same genetic alteration treatments. In all treated sections, morphol- and in situ hybridization results.9 leads to eumelanin overproduction, the time ogy of neoplastic and non-neoplastic cells A recent study demonstrated that a com- required to bleach numerous melanin mole- was maintained. plete bleaching of human melanoma sam- cules is longer than the one required for ples can be reached placing the sections in a fewer molecules.14 Immunohistochemical results 3% H2O2 solution containing 1% for- In feline iris melanoma, neoplastic cell In all tested sample neoplastic cells mamide and irradiating with a 60 W incan- bleaching was achieved with a 1-day light were widely positive for vimentin, S100 descent light bulb.10 However, in this study, exposure. Although a variation in melanin and PNL2, and negative for FVIII and pan- no control sections dipped in the same solu- content between feline and equine neoplas- cytokeratin (Figure 6). Specifically, in all tion but not irradiated were used. Moreover, tic cells cannot be ruled out, it can be also species, atypical cells showed a reticular although the experiment was performed at hypothesized that the difference in the pho- cytoplasmic positivity for vimentin. RT, the temperature reached by the samples, tobleaching time can be explained by a dif- Positivity for S100 ranged from diffuse due to both directly released heat and ener- ferent eu- and pheomelanin content. (swine) to reticular-diffuse (all other gy irradiated by the incandescent light bulb, Specifically, while gray horse dermal species). PNL2 positive reaction had a gran- has not been evaluated. It is in fact known melanoma contains eumelanin, pheome- ular cytoplasmic pattern, more pronounced that more than 80% of the energy radiated lanin, that is known to be more prone to in feline, canine and swine specimens than by incandescent light bulbs is represented photo-degradation than eumelanin, is the in equine sample. On the contrary, while by infrared radiations, invisible to the melanin suspected to be predominant in positivity for FVIII and pancytokeratin was human eye but able to raise the temperature feline iris melanocytes.3,12,14,16 It is interest- detected in the internal positive controls of the irradiated bodies.11 For these reasons ing to note that the same irradiation period (Figure 7), melanocytic neoplastic cells and since a heated 3% H2O2 solution can was able to completely bleach not only neo- resulted negative. determine depigmentation of melanoma plastic pigmented cells but also iris, ciliary histological sections, it cannot be ruled out bodies and retinal pigmented epithelial that the samples tested in that study benefit- cells, derived from the embryonic optical ed of a combined effect of H2O2 and heat- cup of neural ectoderm, producing neu- ing.10 In our study, particular attention was romelanin granules, composed of both eu- Discussion taken in maintaining previously-treated, in- and pheomelanin.2 It can be hypothesized This work aimed to asses if visible light, treatment and control sections in the same that this phenomenon was due to the treat- used to reduce background autofluores- medium and at the same temperature, in ment of 24 h. Shorter treatments could per- cence in fluorescence microscopy, can be order to avoid possible misleading effects haps show more subtle difference in depig- effectively exploited in bright field deriving from variations of these parame- mentation times of the various pigmented microscopy to depigment FFPE melanocyt- ters. Furthermore, the evaluation of the light cell types present in the sample. Obviously, ic neoplasm sections. Indeed, light irradia- source emission spectrum, allowed us to also in this last case it is not possible to tion can permanently alter a molecule exclude a possible effect produced by exclude that the present results are due to a chemical structure causing the loss of its infrared and ultraviolet radiations. For our different proportion of eu/pheo-melanin fluorescence. This phenomenon, named purpose, four pigmented melanocytic neo- content in the pigmented cell populations photobleaching, is usually feared because plasms relatively common in veterinary present in the section. Lack of studies con- capable of reducing the signal of interest. It histopathological routine diagnostic were cerning melanin contents and types in can be instead exploited in order to improve chosen: a feline diffuse iris melanoma, a canine and swine cutaneous melanomas the ratio between signal of interest and non- canine dermal melanoma, a gray horse der- makes it difficult to interpret the results specific signal, damaging endogenous fluo- mal melanoma and a swine cutaneous obtained in these species. Despite this, it is rescent molecules or reducing the adverse melanoma.12-15 Our data demonstrated that a important to underline that in all samples, effects of histological procedures (e.g., auto- complete melanin photo-degradation can be the pigment was completely removed, albeit fluorescence induced by formalin fixation), achieved using a visible-light emitting light with different exposure times. prior to perform an immunofluorescence source, even if different samples required The consistency between the available assay or a fluorescent staining.5-8 The use- different light exposure times. Feline and literature and the results obtained combin- fulness of these protocols is also in the pos- swine sections were completely bleached ing photobleaching with immunohisto- sibility to obtain a targeted effect by damag- following a 1-day treatment, while 2 and 3 chemistry shows that photobleaching does

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Figure 6. Results of the combined photobleaching-immunohistochemical protocol on the feline (A), the canine (B), the equine (C) and the swine (D) specimens. In all tested samples neoplastic cells resulted positive for vimentin (1), S100 (2) and PNL2 (3), and negative for FVIII (4) and pancytokeratin (5). Immunohistochemistry, Mayer's hematoxylin counterstain.

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not affect immunoreactivity of tested mark- buffer pH 7.6 added with 0.5% sodium ers within FFPE samples.12,15,17-21 The poten- azide to prevent bacterial growth. Sodium References tial of this new bleaching method is that, azide can have a dual behavior in photo- compared to traditional chemical bleaching oxidation events, acting either as a strong 1. Solano F. Melanins: Skin pigments and methods, photo-induced bleaching should singlet oxygen quencher or as an azide rad- much more—Types, structural models, offer a greater oxidative selectivity, due to ical or hydroperoxide producer. In particu- biological functions, and formation the use of light at wavelengths preferential- lar it would behave as a pro-oxidant in routes. New J Sci 2014;2014. Doi: ly absorbed by the target molecule, with a acidic environments and as an anti-oxidant 10.1155/2014/498276. hypothetical greater preservation of investi- in neutral environments.29,30 Since TRIS 2. Bush WD, Garguilo J, Zucca FA, gable molecules.5,6,23-25 buffer was the dipping medium in the pres- Albertini A, Zecca L, Edwards GS, et The chemical mechanisms involved in ent experiment, it cannot be ruled out that al. The surface oxidation potential of melanin degradation acts in a sort of auto- sodium azide has slowed down the photo- human neuromelanin reveals a spherical limiting fashion: the oxidation induced by bleaching process quenching the reactive architecture with a pheomelanin core the reactive oxygen species, produced dur- oxygen species produced during melanin and a eumelanin surface. Proc Natl ing melanin photo-irradiation, degrades photo-degradation process. On the con- Acad Sci US 2006;103:14785-9. melanin itself. Consequently, the variation trary, a possible pro-oxidant effect of sodi- 3.Ito S, Wakamatsu K, Sarna T. of the absorption spectrum derived by um azide can be excluded since the dipping Photodegradation of eumelanin and melanin degradation would no longer allow solution was not an acidic medium. pheomelanin and its pathophysiological the formation of reactive oxidizing In conclusion, this pilot study demon- implications. Photochem Photobiol species.3,4 On the contrary, the oxidizing strates that visible light can be effectively 2018;94: 409-20. Doi: 10.1111/ php. effect of solutions routinely used to bleach exploited to completely bleach pigmented 12837. FFPE sections, being absent the targeted melanoma FFPE sections, together with an 4. Szewczyk G, Zadlo A, Sarna M, Ito S, effect ensured by melanin absorption spec- excellent maintenance of cell morphology. Wakamatsu K, Sarna T. Aerobic pho- trum and light source emission spectrum, However, a study involving a larger num- toreactivity of synthetic eumelanins and would continue even after the complete ber of samples is necessary to certainty pheomelanins: generation of singlet melanin degradation. Furthermore, it can be confirm what has been observed in this oxygen and superoxide anion. Pigment hypothesized that a selective effect can also preliminary experiment. Even though this Cell Melanoma Res 2016;29:669-78. be achieved with molecules other than pro- protocol is still not practical for diagnostic Doi: 10.1111/pcmr.12514. teins, such as nucleic acids that could be routine, due to time and special equipment 5. Neumann M, Gabel D. Simple method damaged by H2O2, a chemical oxidant rou- requirements, the obtained results open for reduction of autofluorescence in flu- tinely used to bleach histological sec- interesting perspective on the study of orescence microscopy. J Histochem tions.9,26-28 Although melanin photo-degra- melanin in FFPE samples. In order to Cytochem 2002;50:437-9. Doi: dation involves the production of reactive improve the feasibility of the described 10.1177/002215540205000315. oxygen species, the rapid quench of these protocol, one of the first steps to follow 6. Duong H, Han M. A multispectral LED oxidizing species by melanin itself should would be to reduce the time needed to array for the reduction of background guarantee a lower oxidizing effect on nucle- achieve complete section photobleaching. autofluorescence in brain tissue. J ic acids, compared with the routinely used This achievement could be reached by Neurosci Methods 2013;220:46-54. bleaching solutions.3,4 Thus, photo-induced exploiting different variables such as Doi: 10.1016/j.jneumeth.2013.08.018. bleaching could have a potential use work- incubation temperature, spectral selection 7. Kumar BS, Sandhyamani S, Nazeer SS, ing with in situ hybridization techniques. and energy radiated by incident light, and Jayasree RS. Rapid and simple method In the present study, during photo- the use of a sodium azide-free dipping of photobleaching to reduce back- bleaching, sections were placed in TRIS solution. ground autofluorescence in lung tissue sections. Indian J Biochem Biophys 2015;52:107-10. 8. Sun Y, Ip P, Chakrabartty A. simple elim- ination of background fluorescence in formalin-fixed human brain tissue for immunofluorescence microscopy. J Vis Exp 2017;:e56188. Doi: 10.3791/56188. 9. Schmid FX. Biological macromole- cules: UV-visible spectrophotometry. In S. Robinson, E. Ayres, Editors, Encyclopedia of Life Sciences; J. Wiley & Sons, Hoboken; 2001. Doi: 10.1038 /npg.els.0003142. 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- Figure 7. Canine dermal melanoma. The epidermis stained intensely pancytokeratin pos- 6. Doi: 10.1080/01478885. 2019. itive, neoplastic cells are diffusely negative (A). Blood vessel endothelium was FVIII positive, neoplastic cells were negative (B). Immunohistochemistry, Mayer's hematoxylin 1649886. counterstain. 11. Elvidge CD, Keith DM, Tuttle BT, Baugh KE. Spectral identification of

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Original Paper

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