CIL:38590, 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 21,000 X. See Ladinsky and Howell (2007) for more information on methods used.本研究采用改良的Tokuyasu法（Tokuyasu method）对组织进行免疫电子显微镜样品处理。简言之，将切碎的组织置于含5%蔗糖与100 mM 羟乙基哌嗪乙磺酸（HEPES）的4%多聚甲醛中固定，随后用含2.1 M蔗糖的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）进行浸润处理，持续10小时并多次更换溶液。将固定后的组织转移至铝制冷冻切片标本台（Ted Pella公司，加利福尼亚州雷丁市），随后立即置于液氮中冷冻。使用钻石刀（Diatome品牌），在-110℃条件下用UltraCut UCT/FCS冷冻超薄切片机（Leica公司）切取厚度为90 nm的半薄冷冻切片，并将切片转移至经福尔瓦膜（Formvar）包被、碳镀膜且辉光放电处理的100目铜铑电子显微镜载网。先用含10%胎牛血清的PBS封闭非特异性抗体结合位点后，依次用抗脂肪分化相关蛋白（adipophilin）抗体（豚鼠抗adipophilin抗体）以及反式高尔基体网络糖蛋白38（TGN38）抗体（小鼠单克隆抗体2F7），再结合胶体金标记的二抗（15 nm抗小鼠二抗、10 nm抗豚鼠二抗；Ted Pella公司，加利福尼亚州雷丁市）对切片进行标记，随后用含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型电子显微镜下进行观察，并以数字化方式采集图像，放大倍数为21,000倍。相关实验方法的详细信息可参考Ladinsky与Howell（2007）的研究文献。