Gleiberman, Anatoli (2010) CIL:814, Homo sapiens, permanent cell line cell. CIL. Dataset

This culture of HeLa cells was grown, fixed and labeled directly on 35-mm plastic petri dishes (not on cover slips). To preserve the 3D structure of cells, cultures were fixed in formaldehyde vapors at room temperature over 20 min. Fixation was performed as follows: medium was completely aspirated, a 50ul drop of 37% formaldehyde was placed on the lid of the dish and covered with the dish of cells, bottom up. After fixation dishes were carefully filled with 1ml of 50% glycerol in PBS and dishes were stored at -20 C until staining.

Before staining, cells were incubated with blocking solution (5% fetal calf serum, PBS, 0.2% triton x-100, 25 min room temp.), then treated with primary antibody, which was mouse anti-Cytochrome C from BD Pharmigen (cat.# 556433) diluted 1:100 in blocking solution, 1h room temp. After standard washing (PBS 3-4 times, 5 min each), secondary antibody (donkey monovalent Fab-fragment anti-mouse IgG conjugated with Rhodamine Red-X from Jackson ImmunoResearch laboratories, concentration 2.5ug/ml) was applied.

Images were captured on a Zeiss Axioplan-2. Objective - 63x oil immersion/NA 1.25. Magnifying lens (optovar) was 1.5. Digital camera – Hamamatsu ORCA-ER. Program – AxioVision 3.5.1. Illumination-mercury bulb HBP50. Filter set – standard for Rhodamine/Cy3/Alexa 564 – excitation 530-585 and emission LP 615. Mounting medium-VectaShield.

该HeLa细胞系于35毫米塑料培养皿（非盖玻片）上直接进行培养、固定及标记。为保留细胞的三维结构，培养皿在室温下用福尔马林蒸汽固定超过20分钟。固定过程如下：完全吸除培养基，将50微升37%的福尔马林滴于培养皿盖，并将盛有细胞的培养皿倒置覆盖。固定后，培养皿被小心地填充1毫升50%的甘油PBS溶液，并存储于-20摄氏度直至染色。染色前，细胞与封阻溶液（5%胎牛血清、PBS、0.2%吐温-100，25分钟室温）孵育，随后用一抗处理，一抗为BD Pharmigen公司生产的抗细胞色素C小鼠单克隆抗体（货号556433），以封阻溶液稀释至1:100，室温下处理1小时。在标准洗涤（PBS洗涤3-4次，每次5分钟）后，应用二抗，即Jackson ImmunoResearch实验室生产的单抗抗小鼠IgG Rhodamine Red-X偶联物（浓度2.5微克/毫升）。图像在蔡司Axioplan-2显微镜上捕获，物镜为63倍油浸/NA 1.25，放大镜头（optovar）为1.5倍，数字相机为Hamamatsu ORCA-ER，程序为AxioVision 3.5.1，照明为汞灯HBP50，滤光片组为Rhodamine/Cy3/Alexa 564标准组，激发波长为530-585纳米，发射长波为LP 615，封片介质为VectaShield。