CIL:12912, 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）样品制备。简言之，将切碎的组织置于含5%蔗糖（sucrose）与100 mM HEPES的4%多聚甲醛（paraformaldehyde）中固定，随后用含2.1 M蔗糖的磷酸盐缓冲液（PBS，phosphate-buffered saline）浸润10小时，并多次更换缓冲液。将固定后的组织转移至铝制冷冻切片支架（aluminum cryosectioning stub，Ted Pella, Inc.，加利福尼亚州雷丁市），随即置于液氮中快速冷冻。在-110℃条件下，使用Leica公司UltraCut UCT/FCS冷冻超薄切片机（cryomicrotome）搭配Diatome品牌金刚石刀（diamond knife）切取厚度为90 nm的半薄冷冻切片（semithin cryosections），并将切片转移至经Formvar膜包被、碳蒸镀、辉光放电处理的100目铜铑电子显微镜载网（electron microscopy grid）上。先用PBS配制的10%胎牛血清（calf serum）封闭非特异性抗体结合位点，随后依次孵育抗脂滴包被蛋白（adipophilin）抗体（豚鼠抗adipophilin抗体）与TGN38抗体（小鼠单克隆抗体2F7），再使用胶体金标记二抗（colloidal gold-conjugated secondary antibodies，15 nm抗小鼠二抗、10 nm抗豚鼠二抗，Ted Pella, Inc.，加利福尼亚州雷丁市）完成标记；最后采用含1%乙酸铀酰（uranyl acetate）与1%甲基纤维素（methylcellulose）的双蒸水溶液对切片进行负染色与包埋。样品在Philips CM10型电子显微镜下观察，并以数字化方式采集图像，放大倍数为15500倍。详细实验方法可参考Ladinsky与Howell（2007）发表的相关文献。