PREM images supporting data in figure 4 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9) <br>It contains files for PC12 cells expressing His-tagged Rab3a, Rab27a, Rabphilin3A, Granuphilin-a and Rim2 and tagged with Ni-NTA-gold that are shown in fig. 4a-e.<br><br>HeLa cells were transiently transfected with His-tagged Rab3a, Rab27a, Rabphilin3A, Granuphilin-a and Rim2. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.<br><br>

本数据集隶属于一组成像数据合集（https://doi.org/10.25444/nhlbi.c.5405490），源自Prasai等人2021年发表的题为《神经内分泌细胞中锚定的胞泌致密核心囊泡的纳米级分子形态》的研究论文（https://doi.org/10.1038/s41467-021-24167-9）。<br>本数据集包含表达His标签Rab3a、Rab27a、Rabphilin3A、Granuphilin-a及Rim2，且经Ni-NTA-金标记的PC12细胞相关文件，对应论文图4a-e的实验样本。<br><br>将HeLa细胞瞬时转染带有His标签的Rab3a、Rab27a、Rabphilin3A、Granuphilin-a及Rim2。随后用细胞内缓冲液冲洗细胞，使用2%多聚甲醛，通过19号针头与注射器手动对细胞进行去膜处理。将细胞置于3%牛血清白蛋白（BSA）/磷酸盐缓冲液（PBS）中封闭1小时，再置于经5分钟超声处理的1:5比例10 nm Ni-NTA-纳米金PBS溶液中孵育1小时。采集全内反射荧光（TIRF）成像图后，将样本置于2%戊二醛中固定。后续制备电子显微镜（EM）成像样本：依次经单宁酸与醋酸铀染色、乙醇脱水及临界点干燥处理。将干燥后的样本进行铂/碳复型镀膜，剥离复型膜并转移至电子显微镜载网，使用JEOL 1400电子显微镜及SerialEM免费软件进行图像拼接，以15000倍放大倍率（每像素对应1.2 nm）采集二维透射电子显微镜（TEM）图像。通过二维TEM扫描定位金标记的细胞器，并对对应细胞采集断层扫描数据。以8000倍放大倍率采集单轴倾斜序列（倾角范围-60°至60°，步长1°）。将拼接后的图像进行融合，并使用IMOD软件将倾斜序列重建为三维断层扫描图像。