CIL:12924, 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.组织经改良的Tokuyasu法（Tokuyasu method）处理以用于免疫电子显微镜（immunoelectron microscopy）实验。简言之，将切碎的组织固定于含有5%蔗糖与100 mM HEPES的4%多聚甲醛溶液中，随后用含2.1 M蔗糖的PBS浸润10小时以上，并多次更换溶液。将固定后的组织转移至铝制冷冻切片标本台（Ted Pella, Inc., 加利福尼亚州雷丁市），立即置于液氮中冷冻。使用Leica UltraCut UCT/FCS冷冻切片机（cryomicrotome），搭配Diatome品牌钻石刀，在-110℃条件下切取厚度为90 nm的半薄冷冻切片，将切片转移至经Formvar膜包被、碳涂层处理并辉光放电的100目铜铑合金电子显微镜载网。先用PBS配制的10%小牛血清封闭非特异性抗体结合位点，随后依次使用抗脂肪分化相关蛋白（adipophilin）的抗体（豚鼠抗adipophilin抗体）与抗TGN38的小鼠单克隆抗体2F7，以及胶体金标记的二抗（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）进行孵育标记。之后用含1%乙酸铀酰与1%甲基纤维素的双蒸水溶液进行负染色与包埋。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15,500倍。详细实验方法可参考Ladinsky与Howell（2007）的相关文献。