preparation and alignment of drosophila embryos for injecting fluorescently labeled antibodies

通过抗体介导的实时成像增强蛋白质可视化

Immunofluorescence is a cornerstone technique of modern cell biology originally developed by Albert Coons, which enables the detection of molecules at their native cellular compartments and characterization of the molecular compositions of subcellular organelles or machineries1. Coupled with genetic manipulations, immunofluorescence helps establish the now well-accepted concept that protein localization is essential for its function2. Aside from specific primary antibodies and bright fluorescent dyes, the success of this technique relies on a preliminary process named fixation and permeabilization, which preserves cellular morphologies, immobilizes antigens, and increases the accessibility of antibodies into intracellular compartments. Inevitably, the fixation and permeabilization process would kill cells and terminate all biological processes3. Therefore, immunofluorescence only provides snapshots of the life journey of proteins. However, many biological processes such as cell migration and divisions are dynamic in nature, requiring investigation of protein behaviors in a spatial-temporally resolved manner4,5.
To examine protein dynamics in living organisms, live imaging methods based on genetically encoded fluorescent proteins such as green fluorescent protein (GFP)6 and high-speed confocal microscopes have been developed. Briefly, the protein of interest can be genetically manipulated to be fused with GFP7, and then ectopically expressed from viral or yeast promoters such as cytomegalovirus (CMV)8 or upstream activation sequence (UAS)9. Because GFP is autofluorescent in nature, no fluorophore-coupled antibodies are required to reveal the localization of target proteins, which bypasses the necessity of preliminary processes of fixation or permeabilization. Over the last two decades, fluorescent tags spanning the whole spectrum of wavelength have been developed10, enabling multi-color live imaging of several target proteins at the same time. However, compared to chemically engineered fluorescent dyes such as AlexaFluor or ATTO, the autofluorescence of these genetically encoded fluorescent proteins is relatively weak and unstable when expressed from endogenous promoters, especially during live imaging over longer time scales10. While this shortfall can be mitigated by over-expressing fluorescently tagged target proteins, many with enzymatic activities such as kinases and phosphatases severely disrupt normal biological processes if not expressed at physiological levels.
This protocol presents a method that enables photostable antibody-based target illumination in a live image setup, essentially allowing immunofluorescence without the process of fixation or permeabilization (Figure 1). Through a simple NHS-based primary amine reaction11, one can conjugate fluorescent dyes such as AlexaFluor 488 or 594 with essentially any primary antibody or GFP/HA/Myc nanobody12. Taking advantage of a developmental feature that all Drosophila embryonic cells share a common cytoplasm during the syncytium stage13, one can achieve antigen binding and illumination across entire embryos after the injection of dye-conjugated antibodies. With expanding libraries of endogenously tagged proteins available in Drosophila and other model systems14, this method can potentially broaden applications of these libraries by revealing dynamics of low-abundance fluorescently tagged proteins and other non-fluorescently tagged (HA/Myc-tagged) proteins in living tissues.

作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学 

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

We aim to understand how mechanical and biochemical signals correlate animal morphogenesis, including the detection and the response to forces by cells and molecules. More molecules have been discovered to be mechanosensitive, functioning differently under varying levels of tension. However, the physiological context and the function of these force-sensitive events remain largely unknown.Due to the dynamic nature of force-sensitive events, our field largely relies on high-speed fluorescence imaging and time-lapse recording to capture molecular and cellular behaviors. Many fluorescently-tagged proteins are not bright enough to be imaged, and it requires spatial-temporal resolution without significant bleach. On the other hand, traditional immunofluorescence methods can only capture a snapshot of molecular and cellular fixture after fixation.This method paves the way to dynamically track the localization of many low-abundance protein receptors of major signaling pathways, such as Notch, Wnt, and the BMP pathways. Eventually, in addition to the traditional linear signaling cascades, we can pinpoint and where the signaling events occur at the subcellular scales.

Watch this Scientific Journal Video about Preparation and Alignment of Drosophila  Embryos for Injecting Fluorescently Labeled Antibodies at JoVE.com

Preparation and Alignment of Drosophila  Embryos for Injecting Fluorescently Labeled Antibodies  

用于注射荧光标记抗体的 果蝇 胚胎的制备和比对

首先，将 200 只新孵化的成年果蝇放入塑料圆柱形笼子中。用多孔金属网密封笼子的一侧以通风，另一侧用果汁和酵母糊琼脂板密封。在注射当天，用含有最少酵母糊的果汁板替换笼子的琼脂板。将培养板在25摄氏度下孵育一小时以收集胚胎。然后从笼子中取出盘子以停止产卵，并在 25 摄氏度下再孵育两个小时，以获得两到三个小时的胚胎。要从胚胎中去除蛋壳，请在果汁盘中加入两毫升 50% 漂白剂溶液。用画笔轻轻地将胚胎从培养皿上移开，将培养皿孵育两分钟，同时每30秒旋转一次，以提高蛋壳去除效率。然后将胚胎和漂白剂混合物倒入70微米的尼龙细胞过滤器中。用水瓶全面冲洗胚胎，以消除任何残留的漂白剂和酵母膏。使用干净的剃须刀片，切割矩形琼脂糖块并将块放在载玻片上。用画笔将胚胎从过滤器转移到凝胶块上，并通过轻轻刷洗将它们均匀地分布在块的中心线。现在，准备一个镊子，将镊子的两条腿牢固地粘在一起，形成一个尖锐的尖端。在解剖显微镜下，使用镊子挑选单个胚胎，并沿着凝胶块的延伸边缘对齐它们。在 24 x 50 毫米盖玻片的中点移液 10 微升庚烷胶。使用移液器吸头将胶水涂抹到 0.5 x 3 厘米的矩形区域。使用扁平的镊子，提起涂有胶水的盖玻片并将其稳定地固定在胚胎上方，确保胶水面以大约 45 度面向胚胎。轻轻地将盖玻片压在凝胶块上，让胚胎接触胶水。迅速释放压力并抬高盖玻片。然后在新鲜载玻片的中心分配 10 微升水。将胚胎承载盖玻片放在上面，确保胚胎面朝上。之后，在胚条的一端涂抹 40 微升光晕碳油。调整载玻片的角度，直到光晕碳油覆盖胚胎表面。

Research

JoVE Journal

Developmental Biology

Preparation and Alignment of Drosophila Embryos for Injecting Fluorescently Labeled Antibodies

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

科研

发育生物学

Fluorescent Labeling of Antibodies for Imaging Low Abundance Proteins in Drosophila

Fluorescent Labeling of Antibodies for Imaging Low Abundance Proteins in  Drosophila   

用于果蝇低丰度蛋白成像的抗体荧光标记

Injecting Fluorescently Labeled Antibodies in Live Drosophila Embryos and Imaging of Low Abundance Proteins

Injecting Fluorescently Labeled Antibodies in Live Drosophila  Embryos and Imaging of Low Abundance Proteins  

在活 果蝇 胚胎中注射荧光标记的抗体和低丰度蛋白质的成像