[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提及的ColocZStats工具的性能，我们使用了一项针对DSS1核体与其他类型核体共定位的研究中采集的共聚焦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，其最为关键的功能是参与26S蛋白酶体复合物（26S proteasome complex）介导的蛋白质降解过程。具体实验流程如下：将人卵巢透明细胞癌细胞系RMG-I以每孔10万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，用4%冰浴多聚甲醛在室温（room temperature, RT）下固定细胞15分钟，随后使用含0.1% Triton X-100的0.1%磷酸盐缓冲液（PBSTx）配制的2%牛血清白蛋白（Bovine Serum Albumin, BSA）封闭细胞30分钟。固定与封闭完成后，将细胞与包含抗DSS1抗体（货号NB100-1334，Novus Biologicals公司）和抗PML抗体（货号sc-966，SCBT公司）的一抗混合液在室温下孵育1小时。孵育结束后，用0.1% PBSTx洗涤细胞3次，每次5分钟。随后将细胞与包含山羊抗Alexa Fluor™ 647（用于标记DSS1）、小鼠抗Alexa Fluor™ 488（用于标记PML）以及赫斯特33342（Hoechst 33342，货号H3570，Invitrogen公司）的二抗混合液在室温下孵育1小时。该孵育步骤完成后，再次用0.1% PBSTx洗涤细胞3次，每次5分钟，以确保充分去除未结合的抗体与染料。后续使用搭载Airyscan系统的蔡司LSM800共聚焦显微镜进行Z堆叠成像。本实验采用三种特异性染料分别标记：DSS1核体（红色）、早幼粒细胞白血病（promyelocytic leukemia, PML）核体（绿色）以及细胞核（蓝色）。最终采集的数据文件命名为"示例图像堆叠.tif"（Sample Image Stack.tif）。