背景介绍：

在生理学的广泛背景下，进行体内巨噬细胞特异性去除的能力仍然是一种有效的手段，可以揭示巨噬细胞的功能。与小鼠模型相比，斑马鱼提供了更方便的成像能力，因为它们从单细胞阶段到整个幼虫发育过程中都具有光学透明性。这些特性对于进行体内细胞特异性去除变得非常重要，以便能够实时跟踪和验证目标细胞的消除。斑马鱼中去除巨噬细胞的方法有多种，包括基因去除（例如irf8敲除）、化学遗传学（例如硝基还原酶/甲硝唑系统）和基于毒素的去除（例如使用荷兰Liposoma的氯膦酸盐二钠脂质体Clodronateliposomes）。使用氯膦酸盐二钠脂质体Clodronateliposomes来诱导吞噬脂质体后巨噬细胞凋亡在去除巨噬细胞以及测试它们的吞噬能力方面都是有效的。在这里，我们描述了一种通过静脉注射含荧光右旋糖酐的氯膦酸盐二钠脂质体Clodronateliposomes系统性去除斑马鱼幼虫巨噬细胞的详细方案。与荧光右旋糖酐的共同注射允许实时跟踪巨噬细胞的去除，从验证成功的静脉注射到巨噬细胞对分子摄取及其最终死亡。为了验证巨噬细胞高程度的去除，可以通过在早期幼虫阶段进行氯膦酸盐二钠脂质体Clodronateliposomes注射时快速的中性红活染料染色来确定脑部巨噬细胞（小胶质细胞）的消除程度。

Background：

The ability to conduct in vivo macrophage-specific depletion remains an effective means to uncover functions of macrophages in a wide range of physiological contexts. Compared to the murine model, zebrafish offer superior imaging capabilities due to their optical transparency starting from a single-cell stage to throughout larval development. These qualities become important for in vivo cell specific depletions so that the elimination of the targeted cells can be tracked and validated in real time through microscopy. Multiple methods to deplete macrophages in zebrafish are available, including genetic (such as an irf8 knockout), chemogenetic (such as the nitroreductase/metronidazole system), and toxin-based depletions (such as using clodronate liposomes). The use of clodronate-containing liposomes to induce macrophage apoptosis after phagocytosing the liposomes is effective in depleting macrophages as well as testing their ability to phagocytose. Here we describe a detailed protocol for the systemic depletion of macrophages in zebrafish larvae by intravenous injection of liposomal clodronate supplemented with fluorescent dextran conjugates. Co-injection with the fluorescent dextran allows tracking of macrophage depletion in real time starting with verifying the successful intravenous injection to macrophage uptake of molecules and their eventual death. To verify a high degree of macrophage depletion, the level of brain macrophage (microglia) elimination can be determined by a rapid neutral red vital dye staining when clodronate injection is performed at early larval stages.

氯膦酸二钠脂质体清除斑马鱼巨噬细Protocol解决方案：