A simple and robust protocol for immunostaining Arabidopsis pollen nuclei
A simple and robust protocol for immunostaining Arabidopsis pollen nuclei
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DOI：
10.1007/s00497-018-00360-7
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发表时间：
2019-03-01
影响因子：
3.4
通讯作者：
Berger, Frederic
中科院分区：
文献类型：
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作者：
Borg, Michael;Buendia, Daniel;Berger, Frederic
花粉代表开花植物的雄性性谱系。成熟时，花粉粒由伴体营养细胞和嵌入的精子组成。在花粉发育过程中，这两种细胞类型获得了截然不同的细胞命运。这种差异的命运收购的基础是显着的花粉染色质的重新配置，这是非常明显的细胞学水平。组蛋白标记沉积和命运获取之间的精确联系在很大程度上仍未被探索，这在一定程度上受到了在模式植物物种如拟南芥中处理花粉的困难的阻碍。在这里，我们描述了一个简单而强大的协议，分离拟南芥花粉核和组蛋白标记的免疫染色。除了植物生长，该方案可以在20天内用很少的拟南芥植物进行，从而允许并行分析多个基因型。我们还描述了一种通过抗原修复去除表位掩蔽的方法，该方法极大地改善了针对异染色质组蛋白标记的抗体的信号。
Pollen represents the male sexual lineage in flowering plants. At maturity, pollen grains are composed of a companion vegetative cell with embedded sperm. During pollen development, these two cell types acquire vastly differing cell fates. Underlying this differential fate acquisition is dramatic reconfiguration of pollen chromatin that is highly evident at a cytological level. The precise link between histone mark deposition and fate acquisition remains largely unexplored, which in part has been hindered by the difficulty in working with pollen in model plant species like Arabidopsis. Here, we describe a simple and robust protocol to isolate Arabidopsis pollen nuclei and immunostain for histone marks. Plant growth aside, the protocol can be performed over 2days with few Arabidopsis plants, thus allowing multiple genotypes to be analysed in parallel. We also describe a method to de-mask epitopes through antigen retrieval, which vastly improves the signal for antibodies that target heterochromatic histone marks.