CIL:12929, Rattus rattus, glandular epithelial cell, milk secreting cell, mammary alveolar cell. In Cell Image Library

Tissue was processed for immunoelectron microscopy using a modified Tokuyasu method. Briefly, minced tissue was fixed in 4% paraformaldehyde containing 5% sucrose and 100 mM HEPES and infiltrated with PBS containing 2.1 M sucrose over 10 h, with repeated solution changes. Fixed tissue was transferred to an aluminum cryosectioning stub (Ted Pella, Inc., Redding, CA) and immediately frozen in liquid nitrogen. Semithin (90 nm) cryosections were cut at -110C with an UltraCut UCT/FCS cryomicrotome (Leica), using a diamond knife (Diatome) and transferred to a Formvar-coated, carbon-coated, glow-discharged 100-mesh copper-rhodium electron microscopy grid. Following blocking of nonspecific antibody binding sites with 10% calf serum in PBS, the sections were labeled by sequential incubation with antibodies to adipophilin (guinea pig anti-adipophilin) and TGN38 (mouse monoclonal 2F7) and colloidal gold-conjugated secondary antibodies (15 nm anti-mouse, 10 nm anti-guinea pig; Ted Pella Inc., Redding, CA) and then negatively stained and embedded with 1% uranyl acetate, 1% methylcellulose in distilled water. Samples were viewed in a Philips CM10 electron microscope, and images were collected digitally. Magnification 15,500 X. See Ladinsky and Howell (2007) for more information on methods used.

本研究采用改良Tokuyasu法（modified Tokuyasu method）对组织样本进行免疫电子显微镜（immunoelectron microscopy）处理。简言之，将切碎的组织置于含5%蔗糖与100 mM HEPES的4%多聚甲醛中固定，随后用含2.1 M蔗糖的磷酸盐缓冲液（phosphate-buffered saline, PBS）浸润10小时，期间多次更换缓冲液。将固定后的组织转移至铝制冰冻切片固定桩（Ted Pella, Inc., 加利福尼亚州雷丁市），随后立即投入液氮中冷冻。半薄（90 nm）冰冻切片在-110℃条件下，采用UltraCut UCT/FCS型冰冻切片机（Leica）与钻石刀（Diatome）制备，随后转移至经福尔瓦（Formvar）包被、碳涂层处理并辉光放电的100目铜铑电子显微镜载网。先用含10%胎牛血清的PBS封闭非特异性抗体结合位点，随后依次孵育抗脂滴蛋白（adipophilin）抗体与TGN38（反式高尔基体膜蛋白38）抗体（小鼠单克隆2F7抗体），再使用胶体金标记的二抗（15 nm抗小鼠抗体、10 nm抗豚鼠抗体；Ted Pella Inc., 加利福尼亚州雷丁市）进行标记，最后用含1%乙酸铀酰与1%甲基纤维素的双蒸水进行负染色与包埋。样本在Philips CM10型电子显微镜下进行观察，图像以数字化方式采集，放大倍数为15500倍。详细实验方法可参考Ladinsky与Howell（2007）的研究文献。