Passeron Thierry, Coelho Sergio G, Miyamura Yoshinori, Takahashi Kaoruko, Hearing Vincent J
Pigment Cell Biology Section, Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Exp Dermatol. 2007 Mar;16(3):162-70. doi: 10.1111/j.1600-0625.2006.00538.x.
Although keratinocytes are the most numerous type of cell in the skin, melanocytes are also key players as they produce and distribute melanin that protects the skin from ultraviolet (UV) radiation. In vitro experiments on melanocytic cell lines are useful to study melanogenesis and their progression towards melanoma. However, interactions of melanocytes with keratinocytes and with other types of cells in the skin, such as fibroblasts and Langerhans cells, are also crucial. We describe two techniques, immunohistochemistry (IHC) and tissue in situ hybridization (TISH), that can be used to identify and study melanocytes in the skin and their responses to UV or other stimuli in situ. We describe a practical method to localize melanocytic antigens on formalin-fixed, paraffin-embedded tissue sections and in frozen sections using indirect immunofluorescence with conjugated secondary antibodies. In addition, we detail the use of TISH and its combination with IHC to study mRNA levels of genes expressed in the skin at cellular resolution. This methodology, along with relevant tips and troubleshooting items, are important tools to identify and study melanocytes in the skin.
尽管角质形成细胞是皮肤中数量最多的细胞类型，但黑素细胞也是关键角色，因为它们产生并分布黑色素，保护皮肤免受紫外线（UV）辐射。对黑素细胞系进行的体外实验有助于研究黑色素生成及其向黑色素瘤的发展过程。然而，黑素细胞与角质形成细胞以及皮肤中其他类型细胞（如成纤维细胞和朗格汉斯细胞）的相互作用也至关重要。我们描述了两种技术，免疫组织化学（IHC）和组织原位杂交（TISH），可用于在皮肤中识别和研究黑素细胞及其对紫外线或其他刺激的原位反应。我们描述了一种实用方法，使用偶联二抗的间接免疫荧光法，在福尔马林固定、石蜡包埋的组织切片和冰冻切片上定位黑素细胞抗原。此外，我们详细介绍了TISH的使用及其与IHC的联合应用，以在细胞水平研究皮肤中表达基因的mRNA水平。这种方法以及相关提示和故障排除项目，是识别和研究皮肤中黑素细胞的重要工具。
