Wang Lulu, Chen Yajuan, Niu Di, Tang Mingdong, An Jinjie, Xue Shanshan, Liu Xiaomin, Gao Hongbo
National Engineering Research Center of Tree Breeding and Ecological Restoration, Beijing Forestry University, Beijing 100083, China.
College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China.
Plants (Basel). 2023 Feb 13;12(4):841. doi: 10.3390/plants12040841.
Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements for this method. We found that the stained tissues can be directly observed with a confocal microscope without tissue lysis. Samples maintained at a low temperature (0-4 °C) throughout the process can reduce the intensity of chlorophyll autofluorescence and the background signal. A low temperature is also good for the storage of the sample. Fluorescence signal of the stained samples can be kept for several weeks if they are stored at -20 °C. FtsZ is an essential component of the chloroplast division apparatus. We demonstrated this method with the immunofluorescence staining of FtsZ1 in wildtype Arabidopsis and some chloroplast division mutants. We also successfully tested this method by the immunofluorescence staining of FtsZ1 in many other plants, including woody plants. With these procedures, the performance of tissue-chopping-based immunofluorescence staining method are further improved.
免疫荧光染色是蛋白质亚细胞定位研究中非常常用的方法。一种基于组织切片的叶绿体蛋白免疫荧光染色方法克服了植物细胞壁的限制，使操作更简便，且使用的实验材料更少。在此我们对该方法进行一些改进。我们发现染色后的组织无需组织裂解即可直接用共聚焦显微镜观察。在整个过程中保持低温（0-4°C）的样品可以降低叶绿素自发荧光的强度和背景信号。低温也有利于样品的保存。如果将染色后的样品保存在-20°C，其荧光信号可以保持数周。FtsZ是叶绿体分裂装置的重要组成部分。我们通过在野生型拟南芥和一些叶绿体分裂突变体中对FtsZ1进行免疫荧光染色来证明该方法。我们还通过在许多其他植物（包括木本植物）中对FtsZ1进行免疫荧光染色成功测试了该方法。通过这些步骤，基于组织切片的免疫荧光染色方法的性能得到了进一步提高。
