Mizutani Yuka, Kuga Daisuke, Iida Machiko, Ushida Kaori, Takagi Tsuyoshi, Tokita Yoshihito, Takahashi Masahide, Asai Masato
Division of Perinatology, Institute for Developmental Research, Aichi Human Service Center.
Surgery Department, Anjo Kosei Hospital.
J Vis Exp. 2018 Mar 21(133):57475. doi: 10.3791/57475.
The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 °C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding 'tuft.' Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.
肌动蛋白结合蛋白Girdin是一种胞质蛋白，在各种组织中，肌动蛋白重塑以触发细胞迁移时需要该蛋白。Girdin在酪氨酸1798处被受体酪氨酸激酶和非受体酪氨酸激酶磷酸化。大森等人开发了针对人Girdin酪氨酸1798（pY1798）的位点和磷酸化状态特异性抗体，该抗体特异性结合磷酸化的酪氨酸1798，而不结合未磷酸化的酪氨酸1798。pY1798抗体已用于特异性标记存在于哺乳动物胃肠道组织中的簇状细胞（TCs），但这些细胞的功能尚不清楚。本方案允许使用pY1798抗体和免疫荧光在空肠中对TCs进行强阳性可视化。为确保成功且简单地可视化TCs，本方案包括两种组织学技术：从充满明胶的空肠组织制备游离冷冻切片，以及在50℃下进行3小时的低温抗原修复。用明胶填充空肠可在整个染色过程中保持游离切片的形状，而低温抗原修复可确保来自TCs的强信号。成功使用本方案可使TCs从绒毛顶端到隐窝均呈现pY1798染色。染色的TCs具有线轴状的胞体，荧光信号在管腔顶端聚集，这与突出的“簇”相对应。鬼笔环肽染色与增厚的刷状缘处的pY1798阳性TCs共定位，并且对应于从TC簇延伸的小根团。本方案可用于检查通过胃肠内窥镜收集的人体活检样本中的TCs。此外，最近有报道称，小鼠感染寄生虫后TCs会聚集，这表明本方案可用于诊断人体肠道中的寄生虫感染。
