[Dataset] ColocZStats: A Z-Stack Signal Colocalization Extension Tool for 3D Slicer

To showcase the capabilities of ColocZStats discussed in its manuscript, we utilized confocal z-stack data collected during a study on the colocalization of DSS1 nuclear bodies with other nuclear body types. DSS1, also known as SEM1, is a gene that encodes a protein crucial for various cellular processes, most notably the function of the 26S proteasome complex in protein degradation. More specifically, a human ovarian clear cell carcinoma cell line (RMG-I) was seeded at 100,000 cells per well onto a coverslip in a 6-well plate and allowed to grow till 70\% confluency. On the day of staining, the cells were fixed with 4\% ice-cold paraformaldehyde for 15 mins at room temperature (RT) and blocked with 2\% Bovine Serum Albumin (BSA) in 0.1\% Phosphate Buffer Saline containing 0.1\% Triton-X (PBSTx) for 30 mins. Following fixation and blocking, the cells were incubated with a primary antibody cocktail containing anti-DSS1 (Catalogue\# NB100-1334, Novus Biologicals) and anti-PML (Catalogue\#sc-966, SCBT) for 1h at RT. After that, the cells were washed 3 times with 0.1\% PBSTx for 5 mins each and incubated with a secondary antibody cocktail containing anti-goat Alexa FluorTM 647 (for DSS1), anti-mouse Alexa FluorTM 488 (for PML) and Hoechst 33342 (Catalogue\# H3570, Invitrogen) for 1h at RT. Following this incubation, the cells were subjected to 3 washes, each lasting 5 mins, with 0.1\% PBSTx to ensure thorough cleansing. Subsequently, z-stack imaging was performed using a Zeiss LSM800 confocal microscope with Airyscan. The above process utilized three distinct dyes to specifically label DSS1 nuclear bodies (Red), promyelocytic leukemia (PML) nuclear bodies (Green), and the nucleus (Blue). The data file was named ‘Sample Image Stack.tif’.

为展示其研究论文中论述的ColocZStats工具的性能，我们采用了一项关于DSS1核体与其他核体类型共定位的研究中所采集的共聚焦z堆叠（confocal z-stack）数据。DSS1又称SEM1，是一种编码对多种细胞过程至关重要的蛋白质的基因，其在蛋白质降解过程中，尤其是26S蛋白酶体复合物（26S proteasome complex）的功能发挥方面尤为关键。具体而言，我们将人卵巢透明细胞癌细胞系RMG-I以每孔100,000个细胞的密度接种于6孔板的盖玻片上，培养至细胞汇合度达70%。染色当日，使用4%冰浴多聚甲醛（paraformaldehyde）于室温（RT）固定细胞15分钟，随后用含0.1% Triton-X的0.1%磷酸盐缓冲液（Phosphate Buffer Saline, PBSTx）中的2%牛血清白蛋白（Bovine Serum Albumin, BSA）封闭细胞30分钟。固定与封闭步骤完成后，将细胞与含抗DSS1（货号NB100-1334，Novus Biologicals公司）和抗PML（货号sc-966，SCBT公司）的一抗混合液于室温孵育1小时。孵育结束后，用0.1% PBSTx洗涤细胞3次，每次5分钟；随后将细胞与含抗山羊Alexa Fluor™ 647（用于标记DSS1）、抗小鼠Alexa Fluor™ 488（用于标记PML）以及Hoechst 33342（货号H3570，Invitrogen公司）的二抗混合液于室温孵育1小时。完成二抗孵育后，再次用0.1% PBSTx洗涤细胞3次，每次5分钟以充分去除未结合的试剂。随后，使用搭载Airyscan模块的蔡司（Zeiss）LSM800共聚焦显微镜进行z-stack成像。本实验流程使用三种不同的荧光染料分别标记DSS1核体（红色）、早幼粒细胞白血病（PML）核体（绿色）以及细胞核（蓝色）。最终采集的数据文件命名为"Sample Image Stack.tif"。