National Engineering Research Center of Tree Breeding and Ecological Restoration and State Key Laboratory of Efficient Production of Forest Resources, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, 100083, China.
Plant Cell Rep. 2024 Jun 12;43(7):168. doi: 10.1007/s00299-024-03255-2.
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.
利用植物组织的冰冻切片和巢管进行免疫荧光染色既方便又有效，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不可或缺且广泛应用的技术。目前，使用现有的免疫荧光染色方法，难以有效地观察到叶片中目标蛋白的定位，这些叶片要么坚硬，要么非常薄，要么具有表皮毛或腺体。此外，目标蛋白的信号主要在叶肉细胞中检测到，而不是其他类型的细胞。因此，本研究进一步探索了免疫荧光染色方法的改进。将植物组织用 50%PEG4000 在-60℃下包埋，然后用冷冻切片机制成切片。切片立即浸入固定液中。然后，将样品转移到特殊的嵌套塑料管中，便于固定和免疫荧光染色。该方法使用冰冻切片可以缩短处理时间，减少材料需求。通过优化切片的厚度，可以很好地染色大部分细胞。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，我们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（包括许多叶片坚硬的物种）的保卫细胞和表皮细胞中也观察到了 FtsZ1 的定位。这种方法不仅易于使用，而且还扩大了免疫荧光染色的适用性。
