Xin Xiong, Xin Wang, Ronny Ewanek, Pavan Bhat, Aaron DiAntonio, Catherine A. Collins (2011) CIL:13679, Drosophila melanogaster, motor neuron. CIL. Dataset

The JNK phosphatase puckered, whose expression can be reported via a lacZ enhancer trap, is expressed in very low levels in uninjured neurons; however, nerve crush induces a dramatic increase in puc-lacZ expression in injured motoneurons. This image stack shows puc-lacZ (green) and neuronal nuclei (red) in an uninjured third instar larva. Wandering third instar larvae were dissected in PBS and fixed in either 4% paraformaldehyde in PBS or Bouin’s fixative for 15–30 min. Primary antibodies used were: rat anti-elaV (7E8A10, Developmental Studies Hybridoma Bank) and mouse anti-lacZ (40-1a, DSHB). Secondary antibodies were Alexa 488 anti-mouse and Cy3 anti-rat. Confocal images were collected at room temperature on a spinning-disk confocal system (PerkinElmer) consisting of a scanner (Nipkow CSU10; Yokogawa) and an electron microscopy charge-coupled device camera (C9100-50; Hamamatsu Photonics) mounted on an inverted microscope (Axio Observer; Carl Zeiss, Inc.) with 25× 0.8 NA multi and 40× 1.3 NA, 63× 1.5 NA, and 100× 1.46 NA oil objectives. Similar settings were used to collect all compared genotypes. All imaging and quantification were conducted with Volocity software (PerkinElmer). This image stack corresponds to Fig 1D in J Cell Biol. 2010. 191: 211-223. Images in Fig 1 include CIL#s 13676, 13677, 13678, 13679.

JNK 磷酸酶呈现皱褶状，其表达可通过 lacZ 增强子捕获系统进行检测，在未受伤的神经元中表达水平极低；然而，神经损伤诱导了受伤运动神经元中 puc-lacZ 表达的显著增加。该图像堆栈展示了未受伤的三龄幼虫中 puc-lacZ（绿色）和神经元核（红色）。游走的三龄幼虫在磷酸盐缓冲盐溶液（PBS）中解剖，并固定于磷酸盐缓冲盐溶液中的 4% 甲醛或 Bouin 固定剂中 15-30 分钟。所使用的初级抗体为大鼠抗 elaV 抗体（7E8A10，发育研究杂交瘤库）和抗 lacZ 抗体（40-1a，发育研究杂交瘤库）。二级抗体为 Alexa 488 抗小鼠和 Cy3 抗大鼠。共聚焦图像在室温下使用旋转盘共聚焦系统（PerkinElmer）收集，该系统由扫描仪（Nipkow CSU10；Yokogawa）和电子显微镜电荷耦合器件摄像头（C9100-50；Hamamatsu Photonics）组成，安装于倒置显微镜（Axio Observer；卡尔·蔡司公司）上，配备 25× 0.8 NA 多模和 40× 1.3 NA、63× 1.5 NA、100× 1.46 NA 油镜。所有比较的基因型均采用类似设置进行图像采集。所有成像和量化均使用 Volocity 软件（PerkinElmer）进行。该图像堆栈对应于《细胞生物学杂志》2010年第191期第211-223页的图1D。图1中的图像包括 CIL#s 13676、13677、13678、13679。