David J. Gill, Joanne Chia, Jamie Senewiratne, Frederic Bard (2011) CIL:13564, Homo sapiens, epithelial cell, cervical carcinoma. CIL. Dataset

COP-I beta1 (green) staining at the Golgi (GM130, a cis-Golgi marker) (red) in unstimulated HeLa cells. Helix Pomatia Lectin (HPL) (gray) staining is also shown. HPL binds various glycans but the Tn antigen in particular. The Tn antigen refers to terminal α-linked N-acetyl galactosamine residues (GalNAc) linked to Ser or Thr residues. HeLa cells were serum starved overnight in DME (noFBS). Cells were fixed for 10 min (4% paraformaldehyde) and permeabilized (0.2% Triton X-100). Primary antibody staining followed the manufacturer’s instructions. Cells were subsequently stained for 15–30 min with secondary Alexa Fluor–conjugated antibodies (Alexa 488 for anti-COP-I beta1, Alexa 594 for anti-GM130). Hoechst (blue) and Alexa 647-conjugated-HPL were added during secondary antibody incubations. Cells were mounted onto glass slides using FluorSave (Merck) and imaged at room temperature using an inverted FluoView confocal microscope (model IX81; Olympus) with fluorescence excitation at 488 nm, 561 nm, and 633 nm and either a 60x objective (U Plan Super Apochromatic [UPLSAPO]; NA 1.35) or 100x objective (UPLSAPO; NA 1.40) using Immersol oil. Microscope coupled with a CCD camera (model FVII). Images were acquired and processed using Olympus FV10-ASW software. Image corresponds to Fig 6A, left panel, in J Cell Biol. 189: 843-858. 2010. Images in Fig 6 include CIL#s 13563, 13564, 13565, 13566, 13567, 13568, 13569, 13570.

在未经刺激的HeLa细胞中，对高尔基体（GM130，一种顺式高尔基体标记物）进行COP-I beta1（绿色）染色。同时展示了海螺凝集素（HPL）（灰色）的染色。HPL可与多种糖苷结合，尤其是Tn抗原。Tn抗原指的是连接于丝氨酸或苏氨酸残基的末端α-连接N-乙酰半乳糖胺（GalNAc）残基。HeLa细胞在DME（无FBS）中过夜血清饥饿。细胞固定10分钟（4%多聚甲醛）并透化（0.2%Triton X-100）。一抗染色遵循制造商的说明。随后，细胞与二抗Alexa Fluor偶联抗体（Alexa 488用于抗COP-I beta1，Alexa 594用于抗GM130）染色15-30分钟。在二抗孵育期间加入Hoechst（蓝色）和Alexa 647偶联的HPL。使用FluorSave（默克）将细胞固定在载玻片上，并在室温下使用倒置FluoView共聚焦显微镜（型号IX81；奥林巴斯）进行成像，激发波长分别为488 nm、561 nm和633 nm，使用60x（U Plan Super Apochromatic [UPLSAPO]; NA 1.35）或100x（UPLSAPO; NA 1.40）物镜，并采用Immersol油。显微镜与CCD相机（型号FVII）耦合。图像使用奥林巴斯FV10-ASW软件获取和处理。图像对应于J Cell Biol. 189: 843-858. 2010年图6A左侧面板。图6中的图像包括CIL#s 13563, 13564, 13565, 13566, 13567, 13568, 13569, 13570。