Stack Stephen M, Anderson Lorinda K
Department of Biology, Colorado State University, Fort Collins, CO, USA.
Methods Mol Biol. 2009;558:147-69. doi: 10.1007/978-1-60761-103-5_10.
Many of the structures involved in meiotic synapsis and recombination such as synaptonemal complexes (SCs) and recombination nodules (RNs) can be resolved only by electron microscopy. Therefore, electron microscopic (EM) immunolocalization using gold-conjugated antibodies is the best way to verify whether certain proteins are components of SCs or RNs. Here, we describe (1) preparing tomato primary microsporocyte protoplasts in leptotene, zygotene, and pachytene stages; (2) hypotonically bursting the protoplasts on glow-discharged glass and plastic-coated slides to make spreads of SCs; (3) immunolabeling proteins in SCs and RNs with colloidal gold; (4) staining SC spreads for EM; and (5) transferring SC spreads on plastic films to grids for EM.
许多参与减数分裂联会和重组的结构，如联会复合体（SCs）和重组结节（RNs），只能通过电子显微镜观察到。因此，使用金标抗体的电子显微镜（EM）免疫定位是验证某些蛋白质是否为SCs或RNs组成成分的最佳方法。在此，我们描述了（1）制备处于细线期、偶线期和粗线期的番茄初级小孢子母细胞原生质体；（2）在经辉光放电处理的玻璃片和塑料涂层载玻片上低渗裂解原生质体以制备SCs铺片；（3）用胶体金对SCs和RNs中的蛋白质进行免疫标记；（4）对SCs铺片进行EM染色；以及（5）将SCs铺片转移到塑料薄膜上再置于电镜载网上用于EM观察。
