Melan a

Manufactured by Agilent Technologies

Sourced in Denmark

Melan-A is a protein marker used in immunohistochemistry and flow cytometry applications. It is commonly employed in the identification and characterization of melanocytic lesions, such as melanoma.

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Lab products found in correlation

Hmb45, by Agilent Technologies (4 mentions) Alexa fluor 594 labeled secondary antibody, by Thermo Fisher Scientific (2 mentions) Ki 67 mib1, by Zytomed Systems (2 mentions) Hmb 45, by Cell Marque (1 mentions) Vimentin, by Agilent Technologies (1 mentions) Benchmark xt system, by Roche (1 mentions) Cd31 clone jc70a, by Agilent Technologies (1 mentions) S 100 protein, by Agilent Technologies (1 mentions) Stat6, by Santa Cruz Biotechnology (1 mentions) Cathepsin k, by Abcam (1 mentions) Desmin clone d33, by Agilent Technologies (1 mentions) Alpha smooth muscle actin clone 1a4, by Agilent Technologies (1 mentions) Cd34 clone bi 3c5, by Zytomed Systems (1 mentions) Erg epr3864, by Roche (1 mentions) Ttf1 clone 8g7g3 1, by Zytomed Systems (1 mentions) Anti pax 8, by Cell Marque (1 mentions) Tyrosinase, by Cell Signaling Technology (1 mentions) P erk1 2, by Cell Signaling Technology (1 mentions) Z031129, by Agilent Technologies (1 mentions) Image pro plus version 4, by Media Cybernetics (1 mentions)

13 protocols using melan a

Melanoma Cell Differentiation Protocol

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Available tumor tissue from 47 cases was stained using HMB-45 (Cell Marque, NL) and/or Ki-67 (Spring Bioscience, USA), Melan-A (Dako, DEN) as recommended by the manufacturer and slides were evaluated by two observers (D.S. and M.M.S.S.E.) in order to properly differentiate melanoma cells from nevus cells.

Shitara D., Tell-Martí G., Badenas C., Enokihara M.M., Alós L., Larque A.B., Michalany N., Puig-Butille J., Carrera C., Malvehy J., Puig S, & Bagatin E. (2015). Mutational status of nevus associated-melanomas. The British journal of dermatology, 173(3), 671-680.

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Immunostaining of Melanocytic Markers

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Monolayer cells were fixed with 4% paraformaldehyde and stained with primary antibodies specific for TYR, TYRP1, DCT (polyclonal; a gift from Dr. V.J. Hearing, Bethesda, MD), Melan-A, SILV and S100 (polyclonal; Dako). After washing, cells were incubated with the appropriate Alexa Fluor® 594-labeled secondary antibodies (Invitrogen). Paraffin-embedded slides were deparaffinized, followed by antigen retrieval and staining as described above. Immunofluorescence for DCT was performed as described above. Immunohistochemical staining for S100, Melan-A and Fontana-Masson was performed on paraffin-embedded slides using standard immunoperoxidase techniques. The working solutions of antibodies are shown in Supplementary Table S3.

Yang R., Zheng Y., Li L., Liu S., Burrows M., Wei Z., Nace A., Herlyn M., Cui R., Guo W., Cotsarelis G, & Xu X. (2014). Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors. Nature communications, 5, 5807.

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Histopathological analysis of FFPE samples

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Formalin-fixed, paraffin-embedded (FFPE) tissue samples were obtained. For histopathologic examination, 2 µm-thin sections were routinely stained with hematoxylin-and-eosin. Additional immunohistochemical staining was performed, including e.g., MelanA (1:100, Dako, Glostrup, Denmark; M7196) and Ki-67/MIB1 (1:200, Zytomed, Berlin, Germany; MSK0810). All histologic and IHC sections were reviewed by at least two board-certified dermatopathologists (EH, KG).

Zaremba A., Jansen P., Murali R., Mayakonda A., Riedel A., Krahl D., Burkhardt H., John S., Géraud C., Philip M., Kretz J., Möller I., Stadtler N., Sucker A., Paschen A., Ugurel S., Zimmer L., Livingstone E., Horn S., Plass C., Schadendorf D., Hadaschik E., Lutsik P, & Griewank K. (2022). Genetic and Methylation Analysis of CTNNB1 in Benign and Malignant Melanocytic Lesions. Cancers, 14(17), 4066.

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Comprehensive Immunohistochemistry Panel for Tumor Diagnosis

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Cases were identified in the consultation files of the authors. The tissue specimens were fixed in formalin and processed routinely for histopathology. Immunohistochemistry (IHC) was performed on 3-μm sections cut from paraffin blocks using a fully automated system (“Benchmark XT System”, Ventana Medical Systems Inc., 1910 Innovation Park Drive, Tucson, Arizona, USA) and the following antibodies: vimentin (V9, 1:100, Dako), keratin cocktail (clone AE1/AE3, 1:40, Zytomed, Berlin, Germany), p63 (SSI6, 1: 100, DCS), desmin (clone D33, 1:250, Dako), alpha smooth muscle actin (clone 1A4, 1:200, Dako), HMB45 (clone HMB45, 1:50, Enzo), Melan A (clone A103, 1:50, Dako), CD34 (clone BI-3C5, 1:200, Zytomed), ERG (EPR3864, prediluted, Ventana), CD31 (clone JC70A, 1:20, Dako), S100 protein (polyclonal, 1:2500, Dako), SOX10 (polyclonal, 1:25, DCS), PAX8 (polyclonal rabbit anti-PAX8, 1:50, Cell Marque), NSE (clone BBS/NC/VI-H1, 1:300, Dako), TTF1 (clone 8G7G3/1, 1:500, Zytomed Systems, Berlin, Germany), Napsin A (MRQ-60, ready-to-use, Medac), calretinin (polyclonal, 1:100, Zytomed), alpha-inhibin (clone R1, 1:50, Serotec), GFAP (Clone GFA, 1/1000, DakoPatts, Denmark), EMA (clone E29, 1:200, Dako), STAT6 (clone S-20, 1:1000, Santa Cruz Biotechnology), TFE3 (clone MRQ-37, 1:100, Cell Marque), Cathepsin-K (clone 3F9, 1:50, Abcam) and Ki67 (clone MiB1, 1:100, Dako).

Agaimy A., Stoehr R., Michal M., Christopoulos P., Winter H., Zhang L., Stenzinger A., Michal M., Mechtersheimer G, & Antonescu C.R. (2021). Recurrent YAP1-TFE3 Gene Fusions in Clear Cell Stromal Tumor of The Lung. The American journal of surgical pathology, 45(11), 1541-1549.

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Immunohistochemical Characterization of PDX

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PDX pieces were fixed in formalin and embedded in paraffin. Slides were stained for H&E, S100 (Z031129, DakoCytomation), gp-100 (MS-264-S0, Thermo Scientific), melanA (M719629, DakoCytomation), tyrosinase (T311, 9319, Cell Signaling Technology), and p-ERK1/2 (E10, 4370, Cell Signaling Technology) by our in-house Animal Pathology facility. The NKI-AVL Core Facility Molecular Pathology & Biobanking (CFMPB) provided the NKI-AVL Biobank patient material and performed the BRAF^V600E (VE1, Spring Bioscience) staining according to the manufacturer’s protocol.

Kemper K., Krijgsman O., Kong X., Cornelissen-Steijger P., Shahrabi A., Weeber F., van der Velden D.L., Bleijerveld O.B., Kuilman T., Kluin R.J., Sun C., Voest E.E., Ju Y.S., Schumacher T.N., Altelaar A.F., McDermott U., Adams D.J., Blank C.U., Haanen J.B, & Peeper D.S. (2016). BRAFV600E Kinase Domain Duplication Identified in Therapy-Refractory Melanoma Patient-Derived Xenografts. Cell Reports, 16(1), 263-277.

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Quantitative Analysis of Melanocyte and Immune Cell Markers in Skin Biopsies

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Skin biopsies were obtained from 47 lesional skin of the 47 patients and from nonlesional normal skin of the 5 patients. Hematoxylin and eosin (H&E), Fontana-Masson stainings were performed. Immunohistochemical staining was performed on 4-µm-thick, formalin-fixed, paraffin-embedded tissue sections using antibodies against Melan A (DAKO, Glostrup, Denmark), CD8 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and c-kit+ (CD117, Santa Cruz Biotechnology) in a Ventana autostaining system (Ventana Medical Systems Inc., Tucson, AZ, USA). Image analysis was performed using Image Pro Plus Version 4.5 (Media Cybernetics Co., Silver Spring, MD, USA). The amount of melanin was recorded as the percentage of pigmented area (PA) to measured epidermal area (EA) (PA/EA, ×100, %); Fontana-Masson staining value. Epidermal c-kit+ and CD8 expression was measured as the percentage of stained area (SA) to measured EA (SA/EA, ×100, %); c-kit+ staining value and CD8 staining value, respectively. The number of Melan A+ MC per 1 mm length of the rete ridge (1R) (MC/1R); Melan A staining value.

Park O.J., Han J.S., Lee S.H., Park C.S., Won C.H., Lee M.W., Choi J.H, & Chang S.E. (2016). Expression of Epidermal c-Kit+ of Vitiligo Lesions Is Related to Responses to Excimer Laser. Annals of Dermatology, 28(4), 457-463.

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Immunostaining of Melanocytic Markers

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Histopathological Characterization of Melanocytic Tumors

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Formalin-fixed, paraffin-embedded (FFPE) tissue samples of 19 MT-CNS were
obtained. For histopathologic examination, 2 μm-thin sections were routinely
stained with hematoxylin-and-eosin. Additional immunhistochemical (IHC) stainings for S100
(1:5000, Dako, Glostrup, Danmark; Z0311), melanocytic markers Melan A (1:100, Dako,
Glostrup, Danmark; M7196) and HMB45 (1:200, Dako, Glostrup, Danmark; M0634), BAP1
(described below), and the proliferation marker Ki67/MIB1 (1:200, Zytomed, Berlin,
Germany; MSK0810) were performed.
Diagnoses were made based on criteria described by Brat et al. [23 (link)]. Melanocytomas are well-differentiated
MT-CNS with no or very low mitotic activity (0–1 mitoses per 10 HPF) devoid of CNS
infiltration. Ki67/MIB1-staining is ≤2 %. Intermediate grade melanocytomas
are characterized by increased mitotic activity and microscopic CNS invasion, but are not
sufficiently anaplastic to warrant the designation of malignant melanoma. Ki67/MIB1
staining ranges from 1 to 4 %.
All histologic and IHC sections were reviewed by at least two histopathologists
(JvdN, KGG, MG, TP). The clinico-pathologic details are summarized in Supplemental Table 1.

van de Nes J., Gessi M., Sucker A., Moller I., Stiller M., Horn S., Scholz S.L., Pischler C., Stadtler N., Schilling B., Zimmer L., Hillen U., Scolyer R.A., Buckland M.E., Lauriola L., Pietsch T., Waha A., Schadendorf D., Murali R, & Griewank K.G. (2016). Targeted next generation sequencing reveals unique mutation profile of primary melanocytic tumors of the central nervous system. Journal of neuro-oncology, 127(3), 435-444.

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Immunohistochemical Analysis of Melanoma Markers

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IHC was performed by the department of pathology, FUSCC. To be brief, immunostaining was performed by using an automated BenchMark XT staining system (Ventana Medical Systems, Tucson, AZ, USA) according to manufacturer’s instructions. Proteins were detected using the following antibodies: p16 (1:100, Ventana), AE1/AE3 (1:50, Dako), Ki-67(1:150, Dako), Melan-A (1:50; Dako), HMB45 (1:50, Dako), PNL2 (1:100; MXB Biotechnologies), S-100 (1:700; Dako), SOX10 (1:200; Gene Tech), BRAF (1:50, VE1, Ventana). The antibodies that were chosen to annotate melanoma were recommended from National Comprehensive Cancer Network (NCCN) melanoma guidelines. 3,3'-diaminobenzidine tetrahydrochloride (DAB) was applied to detect the presence of each biomarker in the clinical samples. Finally, the slides were counterstained with hematoxylin for nucleic staining.

Sun W., Zhao F., Hu T., Wu Z., Xu Y., Dong Y., Zheng B., Wang C., Yan W., Zhu X., Wu J., McKay M.J., Arozarena I., Alos L., Teixido C, & Chen Y. (2022). Oncogenic alterations reveal key strategies for precision oncology in melanoma treatment. Annals of Translational Medicine, 10(22), 1246.

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Immunohistochemical Analysis of PRKAR1A, Melan A, and BRAF

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Hematoxylin-eosin and immunohistochemical staining were performed on 4 µm thick sections obtained from formalin-fixed paraffin-embedded tissues (FFPE). The Ventana BenchMark ULTRA system (Ventana, Tucson, USA) was used with the red coloring of the enhanced alkaline phosphatase red detection kit (Ventana; #800-031). The following antibodies were utilized: PRKAR1A (polyclonal rabbit, 1:200 NeoBiotech, Nanterre, France), Melan A (clone A103, 1:50, DAKO, Glostrup, Denmark), BRAF (Clone VE1, 1:100 Spring Bioscience, Pleasanton, CA).

de la Fouchardiere A., Tirode F., Castillo C., Buisson A., Boivin F., Macagno N, & Pissaloux D. (2022). Attempting to Solve the Pigmented Epithelioid Melanocytoma (PEM) Conundrum: PRKAR1A Inactivation Can Occur in Different Genetic Backgrounds (Common, Blue, and Spitz Subgroups) With Variation in Their Clinicopathologic Characteristics. The American journal of surgical pathology, 46(8).

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