CIL:12930, 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 11,500 X. See Ladinsky and Howell (2007) for more information on methods used.

本研究采用改良的Tokuyasu法对组织进行免疫电子显微镜（immunoelectron microscopy）样品制备。简要而言，将切碎的组织置于含有5%蔗糖与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（Phosphate Buffered Saline, PBS）进行10小时的渗透处理，并多次更换缓冲液。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），随即置于液氮中快速冷冻。在-110℃条件下，使用钻石刀（Diatome公司），借助UltraCut UCT/FCS冷冻切片机（Leica公司）切取半薄（90 nm）冷冻切片，随后将切片转移至经Formvar包被、碳镀膜并辉光放电处理的100目铜铑电子显微镜载网。先用含10%胎牛血清的PBS封闭切片上的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）抗体（豚鼠抗adipophilin抗体）、抗反式高尔基体网络蛋白38（TGN38）抗体（小鼠单克隆抗体2F7）以及胶体金标记的二抗（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）对切片进行免疫标记，最后用含1%乙酸铀（uranyl acetate）与1%甲基纤维素（methylcellulose）的双蒸水溶液完成负染色与包埋。样品在飞利浦CM10型电子显微镜下进行观察，并以数字化方式采集图像，放大倍数为11500倍。相关实验方法的详细信息可参考Ladinsky与Howell（2007）的研究文献。