Lee Chanjae, Kieserman Esther, Gray Ryan S, Park Tae Joo, Wallingford John
Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
CSH Protoc. 2008 Feb 1;2008:pdb.prot4957. doi: 10.1101/pdb.prot4957.
INTRODUCTIONImmunocytochemistry (ICC) is widely exploited in studying mammalian systems, but is underutilized among Xenopus developmental biologists. This stems, in part, from the relatively small number of Xenopus antibodies available for use in research. Common misconceptions about ICC in Xenopus embryos also prevail, discouraging researchers from trying the procedure. However, ICC with Xenopus is simple and effective. This article describes methods for whole-mount ICC in Xenopus embryos. Also included are simple procedures to quench autofluorescence of Xenopus and to remove surface pigment from embryos which may interfere with fluorescence imaging. The methods described here are useful for detecting tissue-specific probes (e.g., 12/101 to detect somites). They are also effective for imaging the cytoskeleton (e.g., α-tubulin to detect microtubules) or localizing specific proteins at the subcellular level (e.g., ZO-1 to detect tight junctions). In addition, combining ICC with in situ hybridization is simple and highly effective.
引言
免疫细胞化学（ICC）在研究哺乳动物系统中得到广泛应用，但在非洲爪蟾发育生物学家中却未得到充分利用。这部分源于可用于研究的非洲爪蟾抗体数量相对较少。关于非洲爪蟾胚胎ICC的常见误解也很普遍，这使得研究人员不愿尝试该方法。然而，非洲爪蟾的ICC操作简单且有效。本文介绍了非洲爪蟾胚胎全胚胎免疫细胞化学的方法。还包括淬灭非洲爪蟾自发荧光以及去除可能干扰荧光成像的胚胎表面色素的简单步骤。这里描述的方法可用于检测组织特异性探针（例如，用12/101检测体节）。它们也可有效地用于细胞骨架成像（例如，用α-微管蛋白检测微管）或在亚细胞水平定位特定蛋白质（例如，用ZO-1检测紧密连接）。此外，将免疫细胞化学与原位杂交相结合既简单又高效。
