CIL:12911, 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.

采用改良的Tokuyasu方法处理组织，以开展免疫电子显微镜（immunoelectron microscopy）实验。简而言之，将切碎的组织固定于含有5%蔗糖与100 mM HEPES的4%多聚甲醛（paraformaldehyde）中，随后使用含2.1 M蔗糖的磷酸盐缓冲液（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冰冻切片支架（Ted Pella, Inc., 加利福尼亚州雷丁市），立即置入液氮中冷冻。使用Leica UltraCut UCT/FCS冰冻超薄切片机，搭配Diatome公司的金刚石刀（diamond knife），于-110℃条件下切取厚度为90 nm的半薄切片，将切片转移至经Formvar包被、碳涂层并辉光放电处理的100目铜铑电子显微镜载网。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，随后依次以脂滴蛋白（adipophilin）抗体（豚鼠抗adipophilin）、TGN38抗体（小鼠单克隆2F7）以及胶体金标记的二抗（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）进行孵育标记。之后使用含1%乙酸铀（uranyl acetate）与1%甲基纤维素（methylcellulose）的双蒸水溶液对切片进行负染色并包埋。样品在Philips CM10电子显微镜下观察，以数字化方式采集图像，放大倍数为15,500倍。相关实验方法细节可参阅Ladinsky与Howell（2007）的文献。