IMPROVEMENTS IN IMMUNOSTAINING SAMPLES EMBEDDED IN METHACRYLATE - LOCALIZATION OF MICROTUBULES AND OTHER ANTIGENS THROUGHOUT DEVELOPING ORGANS IN PLANTS OF DIVERSE TAXA
IMPROVEMENTS IN IMMUNOSTAINING SAMPLES EMBEDDED IN METHACRYLATE - LOCALIZATION OF MICROTUBULES AND OTHER ANTIGENS THROUGHOUT DEVELOPING ORGANS IN PLANTS OF DIVERSE TAXA
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DOI：
10.1007/bf00195665
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发表时间：
1992-06-01
期刊：
影响因子：
4.3
通讯作者：
GUBLER, F
中科院分区：
文献类型：
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作者：
BASKIN, TI;BUSBY, CH;GUBLER, F
微管在植物生长发育中起着重要作用。在切片材料中定位微管是有利的，因为它允许对任何感兴趣的组织进行研究，并且允许知道器官内细胞的位置关系。我们在这里描述了一种方法，将半薄（0.5-2 μ m）的材料嵌入甲基丙烯酸丁酯中，并在其中添加10 mM的二硫苏糖醇。去除包埋材料后，采用间接免疫荧光染色，我们获得了微管、肌动蛋白微丝、胼胝体和脉冲输注的溴脱氧尿嘧啶的清晰图像。该方法适用于拟南芥（L.）的根组织。海恩，白松，唐，紫杉树。凤仙花；对湿法蕈菌孢子组织的研究。一般来说，所研究的器官中的大多数细胞都能成功染色。使用这种方法，我们发现所有这些物种的间期分生细胞不仅在通常的皮质阵列中而且在整个细胞质中都有微管。钙螯合剂乙二醇-双（β -氨基乙醚）N， N，N‘，N’-四乙酸（EGTA）在固定缓冲液中的存在导致一定程度的组织损伤，并没有增强微管的保存。含有egta的缓冲液在固定过程中稳定植物微管的普遍假设似乎是没有根据的。
Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5-2-mu-m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana (L.) Heynh, Pinus radiata D. Don, Zamia furfuracea Ait., Azolla pinnata R. Br. and on sporophytic tissues of Funaria hygrometrica Hedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)N, N,N',N'-tetraacetic acid (EGTA) in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.