Electron and immunoelectron microscopy

Intracellular parasites were fixed at room temperature for 1h in 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde. Then, samples were kept overnight at 4°C. After washing with the same buffer, samples were fixed again in aqueous 1% osmium tetroxide and 1.5% potassium ferrocyanide. After successive dehydration in ethanol, samples were embedded in Epon (Agar Scientific, AGR1165): 2h in resin mixed with ethanol and 2h with pure resin before polymerization 24h at 60°C. Sections of 70 nm were cut using an ultramicrotome LEICA UC6, collected on grids, and contrasted 15min in 2% aqueous uranyl acetate and 2min in Reynold’s lead citrate. Ultrastructural images were collected using a JEOL1400 transmission electron microscope at 80kV and camera RIO9. For immuno-electron microscopy, the HFF cells infected with iKO-TgREMIND mutants treated with ATc for 36 hours or untreated were fixed overnight at 4°C in 8% paraformaldehyde with 0.1% glutaraldehyde in PBS buffer, thoroughly washed in the same buffer, pelleted in 10% gelatin and infused overnight in sucrose 2.3 M containing 20% polyvinyl pyrrolidone 10000 in phosphate buffer 0.1 M. The pellets were mounted on ultracryomicrotome supports and rapidly frozen in liquid nitrogen. Ultrathin sections of about 90–100 nm were obtained using a UC6 ultramicrotome equipped with an FC6 device (Leica). For immuno-labeling, sections were incubated in blocking medium (0.05 M glycine, 5% fish gelatine in 0.1 M PBS buffer) for 30 min. The grids were incubated with rabbit polyclonal anti-ROP1 or monoclonal anti-GRA1 antibodies for 1.5 hour at 37°C. After washing, sections were incubated at room temperature for 30 min in the corresponding secondary gold conjugates (Aurion, 10nm gold-labeled antibody) diluted in the same buffer. Following a final thorough wash in PBS alone, the grids were fixed in 4% glutaraldehyde for 10 min at room temperature and washed in water. After staining 0.5% uranyl acetate in 1.5% methylcellulose, sections were observed on a JEOL1400 transmission electron microscope at 80 kV accelerating voltage.

电子和免疫电子显微镜
细胞内寄生虫在含有 2.5% 戊二醛的 0.1 M 二甲胂酸钠缓冲液中室温固定 1 小时。 然后，将样品在 4°C 下保存过夜。 用相同的缓冲液洗涤后，将样品再次固定在1%四氧化锇和1.5%亚铁氰化钾水溶液中。 在乙醇中连续脱水后，将样品包埋在 Epon (Agar Scientific, AGR1165) 中：在与乙醇混合的树脂中嵌入 2 小时，在纯树脂中嵌入 2 小时，然后在 60°C 下聚合 24 小时。 使用超薄切片机 LEICA UC6 切割 70 nm 的切片，收集在网格上，并在 2% 醋酸双氧铀水溶液中对比 15 分钟，在雷诺柠檬酸铅中对比 2 分钟。 使用 80kV 的 JEOL1400 透射电子显微镜和 RIO9 相机收集超微结构图像。 对于免疫电子显微镜，将用 ATc 处理 36 小时或未处理的 iKO-TgREMIND 突变体感染的 HFF 细胞在 8% 多聚甲醛和 0.1% 戊二醛的 PBS 缓冲液中于 4°C 固定过夜，在相同缓冲液中彻底洗涤，沉淀 在 10% 明胶中，并在含有 20% 聚乙烯吡咯烷酮 10000 的 0.1 M 磷酸盐缓冲液的蔗糖 2.3 M 中输注过夜。将沉淀安装在超低温切片机支架上并在液氮中快速冷冻。 使用配备 FC6 装置（徕卡）的 UC6 超薄切片机获得约 90-100 nm 的超薄切片。 对于免疫标记，切片在封闭介质（0.05 M 甘氨酸、0.1 M PBS 缓冲液中的 5% 鱼明胶）中孵育 30 分钟。 将网格与兔多克隆抗 ROP1 或单克隆抗 GRA1 抗体在 37°C 下孵育 1.5 小时。 清洗后，将切片在用相同缓冲液稀释的相应二级金缀合物（Aurion，10nm金标记抗体）中室温孵育30分钟。 最后仅用 PBS 彻底洗涤后，将网格在室温下于 4% 戊二醛中固定 10 分钟并用水洗涤。 用1.5%甲基纤维素中的0.5%乙酸双氧铀染色后，在JEOL1400透射电子显微镜上观察切片。