Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos
Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos
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DOI：
10.3791/59344
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发表时间：
2019-05-01
影响因子：
1.2
通讯作者：
Shive, Heather R.
中科院分区：
文献类型：
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作者：
Ferguson, Jordan L.;Shive, Heather R.
细胞间相互作用的研究通常需要对特定细胞群进行离散标记和精确的蛋白质定位。斑马鱼胚胎是用体内模型检查这种相互作用的绝佳工具。整体免疫组织化学和免疫荧光测定经常应用于斑马鱼胚胎来评估蛋白质表达。然而，在三维空间中实现共定位蛋白质的精确映射可能很困难。此外，一些研究可能需要使用与同一技术不兼容的两种抗体（例如，抗体 1 仅适用于免疫组织化学，抗体 2 仅适用于免疫荧光）。本文描述的方法的目的是对源自早期斑马鱼胚胎的个体冰冻切片进行连续免疫荧光和/或免疫组织化学。在这里，我们描述了使用连续轮次的免疫荧光、成像、免疫组织化学、单次冰冻切片成像，以实现单细胞水平上蛋白质表达的精确鉴定。该方法适用于任何需要准确识别单个细胞中多个蛋白质靶标的早期斑马鱼胚胎研究。
Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.