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Practical methods for detecting melanocytes in the skin and their responses to UV
Author(s) -
Coelho Sergio,
Passeron Thierry,
Miyamura Yoshinori,
Hearing Vincent J
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a774-c
Subject(s) - melanin , melanocyte , immunohistochemistry , human skin , skin cancer , in situ hybridization , melanoma , antigen , biology , antigen retrieval , in vivo , antibody , monoclonal antibody , immunofluorescence , pathology , immunology , cancer research , gene expression , medicine , cancer , gene , biochemistry , genetics , microbiology and biotechnology
Recently, there has been significant literature and discussion about the effects of ultraviolet (UV) exposure on the human body. Pigmentation provided by melanin protects us from harmful effects of UV exposure (including skin cancer). Although keratinocytes are the most numerous type of cell in the skin, melanocytes are also key players since they produce and distribute melanin to protect the skin from UV radiation. In vitro experiments on melanocytic cell lines are helpful to study melanogenesis and the progression of melanocytes toward melanoma. However, interactions of melanocytes not only with keratinocytes but also with other types of cells in the skin, such as fibroblasts and Langerhans cells, also appear to be crucial. Using two techniques, immunohistochemistry and tissue in situ hybridization (TISH), we have optimized methods to identify and study melanocytes in the skin and their responses to UV in vivo . We describe a practical method to localize melanocytic antigens on formalin‐fixed, paraffin‐embedded tissue sections and frozen sections using indirect immunofluorescence with conjugated secondary antibodies. We also show a comparison of the effects of two methods of antigen retrieval to optimize staining of our melanocyte markers. In addition, we discuss the combination of immunohistochemistry with tissue in situ hybridization to the study mRNA levels of genes in the skin responding to UV. The importance of the TISH technique is underscored by the fact that it permits us to study expression of transcription factors involved in melanogenesis, to which antibodies are not available. Our methodology along with relevant tips and troubleshooting items are important tools in understanding melanocyte responses to UV.