Three-colour fluorescence immunohistochemistry reveals the diversity of cells staining for macrophage markers in murine spleen and liver
Three-colour fluorescence immunohistochemistry reveals the diversity of cells staining for macrophage markers in murine spleen and liver
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DOI：
10.1016/j.jim.2008.02.005
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发表时间：
2008-05-20
影响因子：
2.2
通讯作者：
Dunbar, P. Rod
中科院分区：
文献类型：
--
作者：
Lloyd, Catherine M.;Phillips, Anthony R. J.;Dunbar, P. Rod
传统上使用小范围的标记物（通常是 F4/80、CD68 和 CD11b）在小鼠组织中鉴定巨噬细胞。然而，许多研究表明巨噬细胞群体存在显着的异质性，并且没有单个标记，甚至一对标记，可以必然识别所有群体。此外，许多关键的单克隆抗体是在同一物种中产生的，因此很难在组织化学研究中将它们组合起来。在这里，我们利用抗 FITC 技术优化了三色免疫荧光染色方案，以允许同时使用巨噬细胞标记物的抗体。我们强调正常肝脏和脾脏中 F4/80、CD68、CD11b 和 CD11c 染色细胞的显着异质性。使用饮食诱导的脂肪性肝炎作为肝脏炎症模型，我们发现 CD11b 由新迁移的巨噬细胞前体细胞表达，但单独使用时，它是巨噬细胞前体细胞的不可靠标记，因为它也由迁移的中性粒细胞表达。在健康肝脏中，CD11c 表达是紧邻血窦周围细胞群的独特特征。然而，在肝脏炎症过程中，CD11c 也可以与其他细胞共表达，包括肝实质内的浸润细胞和 F4/80(+) 细胞。虽然没有一种标记物足以解释所有巨噬细胞群体，但我们确认 F4/80 标记了肝脏和脾脏中的大多数组织驻留巨噬细胞，尽管也存在 CD68、CD11b 或 CD11c 阳性的 F4/80(-) 群体。用这些标记区分组织巨噬细胞和树突细胞仍然存在问题。 (C) 2008 Elsevier B.V. 保留所有权利。
Macrophages have traditionally been identified in murine tissues using a small range of markers, typically F4/80, CD68 and CD11b. However many studies have suggested that substantial heterogeneity exists in macrophage populations, and no single marker, nor even pair of markers, can necessarily identify all the populations. Further, many of the key monoclonal antibodies have been raised in the same species, making it difficult to combine them in histochemical studies. Here we have optimised a triple colour immumofluorescent staining protocol, utilising an anti-FITC technique, to allow antibodies to macrophage markers to be used simultaneously. We highlight the substantial heterogeneity of cells in both normal liver and spleen that stain for F4/80, CD68, CD11b, and CD11c. Using diet-induced steatohepatitis as a model of liver inflammation, we show that CD11b is expressed by newly migrating macrophage precursors, but is an unreliable marker for macrophage precursors when used alone because it is also expressed by migrating neutrophils. In healthy livers CD11c expression is a unique feature of a population of cells immediately surrounding the sinusoids. However, during hepatic inflammation CD11c can also be co-expressed by other cells, including both infiltrating cells and F4/80(+) cells within the liver parenchyma. While no one marker alone is sufficient to account for all macrophage populations, we confirm that F4/80 marks the majority of the tissue-resident macrophages in both the liver and the spleen, although F4/80(-) populations that are positive for CD68, CD11b, or CD11c also exist. Distinguishing between tissue macrophages and dendritic cells with these markers remains problematic. (C) 2008 Elsevier B.V. All rights reserved.