CIL:12931, 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.

采用改良Tokuyasu法（Tokuyasu method）开展组织的免疫电子显微镜（immunoelectron microscopy）处理。具体操作简述如下：将切碎的组织固定于含5%蔗糖（sucrose）与100 mM HEPES的4%多聚甲醛（paraformaldehyde）中，随后用含2.1 M蔗糖的磷酸盐缓冲液（PBS）浸润10小时以上，并多次更换缓冲液。将固定完成的组织转移至铝制冷冻切片标本托（aluminum cryosectioning stub，Ted Pella, Inc., 雷丁, 加利福尼亚州），即刻置入液氮（liquid nitrogen）中快速冷冻。使用Leica UltraCut UCT/FCS冷冻切片机（cryomicrotome），搭配Diatome品牌钻石刀（diamond knife），于-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包被、碳蒸镀及辉光放电处理的100目铜铑电子显微镜载网。先用含10%胎牛血清（calf serum）的PBS封闭切片的非特异性抗体结合位点，随后依次孵育抗脂滴蛋白（adipophilin）抗体（豚鼠来源抗adipophilin抗体）与TGN38抗体（小鼠单克隆抗体2F7），再使用胶体金标记的二抗（colloidal gold-conjugated secondary antibodies，15 nm羊抗小鼠抗体、10 nm羊抗豚鼠抗体；Ted Pella Inc., 雷丁, 加利福尼亚州）完成标记，最后用含1%乙酸铀（uranyl acetate）与1%甲基纤维素（methylcellulose）的双蒸水溶液进行负染色与包埋。样品在Philips CM10电子显微镜下完成观察，并以数字化方式采集图像，放大倍数为11,500倍。相关实验方法的详细信息可参阅Ladinsky与Howell（2007）的文献。