Sungsu Kim, Yogesh P. Wairkar, Richard W. Daniels, Aaron DiAntonio (2011) CIL:13474, Drosophila melanogaster, motor neuron. CIL. Dataset

Colocalization between DVGLUT (red) aggregates and the endosomal ESCRT protein Hrs (green, also known as Vps27) in motoneuron cell bodies of the ventral nerve cord of a wild-type larva. Wandering third instar larvae were dissected in ice-cold PBS and fixed with Bouin’s solution for 5 min. Blocking and antibody incubation were performed in PBS containing 0.1% Triton X-100. anti-DVGLUT (Daniels et al., 2004) antibody was used at 1: anti-HRS-FL (Lloyd et al., 2002) was used at 1:1000. Secondary antibodies (Alexa 488-/Cy3-conjugated) were used at 1:1000. After staining, specimens were equilibrated in 70% glycerol in PBS and mounted with VectaShield (Vector Laboratories). Confocal images were acquired with a confocal microscope (model C1; Nikon) and accompanying EZ-C1 software using argon (excitation at 488 nm) and HeNe (excitation at 543 and 633 nm) lasers and a 60x Plan-Apochromat NA 1.4 objective (Nikon) at room temperature. Samples for each experiment were processed using the same confocal gain setting. Image corresponds to Figure 2A, top 3 panels in Kim et al. J Cell Biol. 188: 717-734. 2010.

在野生型幼虫腹神经节运动神经元细胞体内，DVGLUT（红色）聚集体与内体ESCRT蛋白Hrs（绿色，亦称Vps27）的共定位。对游走性的第三龄幼虫进行冰冷的PBS溶液解剖，并使用Bouin溶液固定5分钟。在含有0.1% Triton X-100的PBS中进行封闭和抗体孵育。使用1:比例的anti-DVGLUT（Daniels等人，2004）抗体，以及1:1000比例的anti-HRS-FL（Lloyd等人，2002）抗体。采用1:1000比例的Alexa 488-/Cy3偶联的二级抗体。染色后，样品在含有70%甘油和PBS的溶液中平衡，并使用Vector Laboratories的VectaShield进行封片。使用型号为C1的共聚焦显微镜（Nikon）及其配套的EZ-C1软件，在室温下，通过氩激光（激发波长为488 nm）和HeNe激光（激发波长为543和633 nm）以及60x Plan-Apochromat NA 1.4物镜（Nikon）获取共聚焦图像。每个实验的样品均使用相同的共聚焦增益设置进行处理。图像对应于Kim等人《细胞生物学杂志》第188卷第717-734页（2010年）的图2A顶部3个面板。