Yan Shan Ong, Bor Luen Tang, Li ShenLoo, Wanjin Hong (2011) CIL:13647, Homo sapiens. CIL. Dataset

This image is Fig. 4B from PMID: 20679433.

HeLa cells were fixed and double-labeled with rabbit anti-p125A (secondary Ab: goat anti-rabbit Ab conjugated with Alexa 555, shown in red) and mouse anti-Sec31A (secondary Ab: goat anti-mouse Ab conjugated with FITC, shown in green) antibodies.

Technical details: Cells grown on coverslips were washed 2X with PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 (PBSCM), fixed with 4% paraformaldehyde in PBSCM for 20 min at RT, washed 5X at 5-min intervals using PBSCM, and then permeabilized with 0.1% Saponin in PBSCM for 20 min at RT. The cells were then immunolabeled with appropriate primary antibodies diluted in fluorescence dilution buffer (FDB; PBSCM with 5% FBS and 2% bovine serum albumin [BSA]) for 1 h at RT, washed 5X with 0.1% Saponin PBSCM at 5-min intervals. Secondary antibodies were diluted in FDB and incubated at RT for 1 h, washed again with 0.1% Saponin PBSCM 5X at 5-min intervals, and then 2X with PBSCM. The coverslips were mounted on microscopic slides with Vectashield mounting medium containing DAPI. Confocal microscopy was performed with an Axioplan II microscope (Carl Zeiss, Inc.) equipped with Zeiss confocal scanning optics. 100x objective.

本图源自PMID: 20679433的参考文献，呈现为图4B。HeLa细胞经固定处理后，采用兔抗p125A抗体（二抗为用Alexa 555标记的山羊抗兔抗体，以红色显示）和鼠抗Sec31A抗体（二抗为用FITC标记的山羊抗鼠抗体，以绿色显示）进行双重标记。技术细节如下：将培养在盖玻片上的细胞用含有1 mM CaCl2和1 mM MgCl2的磷酸盐缓冲液（PBSCM）洗涤两次，在室温下用4%多聚甲醛固定于PBSCM中20分钟，并以5分钟间隔用PBSCM洗涤5次，随后用0.1%皂素在PBSCM中室温处理20分钟以通透化细胞。接着，将细胞用适当的一抗在荧光稀释缓冲液（FDB；含有5%胎牛血清和2%牛血清白蛋白的PBSCM）中稀释，室温下孵育1小时，并以0.1%皂素PBSCM在5分钟间隔内洗涤5次。二抗用FDB稀释并在室温下孵育1小时，再次用0.1%皂素PBSCM在5分钟间隔内洗涤5次，然后用PBSCM洗涤两次。将盖玻片用含有DAPI的VectorShield封片剂固定在显微镜载玻片上。使用配备蔡司共聚焦扫描光学系统的Axioplan II显微镜（蔡司公司）和100倍物镜进行共聚焦显微镜检查。