Abstract：
CONCLUSIONS: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.
摘要：
结论:植物组织冰冻切片和巢管免疫荧光染色简便有效,拓宽了免疫荧光染色的适用性。免疫荧光染色是确定叶绿体分裂蛋白亚细胞定位的必不可少且广泛使用的技术。目前,很难有效地观察靶蛋白在硬叶中的定位，或者非常薄，或有表皮毛发或腺体用目前的免疫荧光染色方法。此外，靶蛋白信号主要在叶肉细胞中检测到，不是其他类型的细胞。因此，本研究进一步探索免疫荧光染色方法的改进。植物组织在-60℃下用50%PEG4000包埋，然后用冷冻切片机切成几段。立即将切片浸入固定溶液中。然后，样品被转移到一个特殊的嵌套塑料管中，这有利于固定和免疫荧光染色程序。在这种方法中使用冷冻切片可以缩短处理时间并减少材料需求。通过优化截面的厚度，大部分细胞可以被很好地染色。使用这种方法，我们观察到叶绿体分裂蛋白FtsZ1在野生型拟南芥和各种叶绿体分裂突变体中的定位。同时,不仅在叶肉细胞中观察到FtsZ1的定位，而且在许多其他植物物种的保卫细胞和表皮细胞中，包括许多具有硬叶组织的物种。这种方法不仅易于使用，而且扩大了免疫荧光染色的适用范围。
