Ferguson Jordan L, Shive Heather R
J Vis Exp. 2019 May 14(147). doi: 10.3791/59344.
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.
细胞间相互作用的研究通常需要对特定细胞群体进行离散标记以及精确的蛋白质定位。斑马鱼胚胎是利用体内模型研究此类相互作用的极佳工具。全胚胎免疫组织化学和免疫荧光检测常用于斑马鱼胚胎以评估蛋白质表达。然而，在三维空间中实现共定位蛋白质的精确映射可能具有挑战性。此外，一些研究可能需要使用两种不兼容同一技术的抗体（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）。本文所述方法的目的是对源自早期斑马鱼胚胎的单个冷冻切片进行连续免疫荧光和/或免疫组织化学检测。在此我们描述了对单个冷冻切片进行连续多轮免疫荧光、成像、免疫组织化学、成像，以便在单细胞水平上精确鉴定蛋白质表达。该方法适用于任何需要在早期斑马鱼胚胎中精确鉴定单个细胞内多个蛋白质靶点的研究。
