CIL:12919, 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 28,500 X. See Ladinsky and Howell (2007) for more information on methods used.采用改良的Tokuyasu法（modified Tokuyasu method）对组织进行免疫电子显微镜术（immunoelectron microscopy）处理。简言之，将切碎的组织固定于含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冷冻切片托（Ted Pella, Inc.，加利福尼亚州雷丁市），并立即置于液氮（liquid nitrogen）中快速冷冻。使用钻石刀（diamond knife），在-110℃条件下通过Leica UltraCut UCT/FCS冷冻超薄切片机（cryomicrotome）切取厚度为90 nm的半薄切片，将切片转移至经Formvar膜（Formvar）包被、碳蒸镀并经辉光放电（glow-discharged）处理的100目铜铑电子显微镜载网（copper-rhodium electron microscopy grid）。先用含10%胎牛血清的PBS封闭非特异性抗体结合位点（nonspecific antibody binding sites），随后依次对切片进行孵育标记：先使用豚鼠抗脂肪分化相关蛋白（adipophilin）抗体，再使用小鼠单克隆抗体2F7（针对反式高尔基体网络蛋白38（TGN38）），最后使用胶体金标记的二抗（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）。完成标记后，使用含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（negative staining）并包埋。样品在Philips CM10型电子显微镜（electron microscope）下观察，以数字化方式采集图像，放大倍数为28500倍。详细实验方法可参考Ladinsky与Howell（2007）的相关文献。