INSITU ENZYME-HISTOCHEMISTRY ON PLASTIC-EMBEDDED PLANT-MATERIAL - THE DEVELOPMENT OF AN ARTIFACT-FREE BETA-GLUCURONIDASE ASSAY
INSITU ENZYME-HISTOCHEMISTRY ON PLASTIC-EMBEDDED PLANT-MATERIAL - THE DEVELOPMENT OF AN ARTIFACT-FREE BETA-GLUCURONIDASE ASSAY
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DOI：
10.1111/j.1365-313x.1992.00261.x
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发表时间：
1992-03-01
期刊：
影响因子：
7.2
通讯作者：
DEBROUWER, D
中科院分区：
文献类型：
--
作者：
DEBLOCK, M;DEBROUWER, D
描述了一种原位酶组织化学方法，可以保留组织和细胞结构以及酶活性。来自野生型和转基因甘蓝型油菜植物的处于不同发育阶段的花芽被用作起始材料，后者含有受绒毡层特异性启动子控制的GUS基因。该方法基于以下原理：在碎冰上处理组织，不固定，但用亚精胺预处理，用丙酮部分脱水，最后在 5 ℃ 下包埋在与水混溶的甲基丙烯酸乙二醇酯树脂中。该方法用于建立灵敏的高分辨率β-葡萄糖醛酸酶组织化学测定。琥珀酸脱氢酶测定作为组织和细胞活力的对照以及不同组织和细胞类型之间相对代谢活动的标准。
An in-situ enzyme histochemical method is described that preserves the tissue and cell structure as well as the enzyme activities. Flower buds at different developmental stages from wild-type and transgenic Brassica napus plants, the latter containing the GUS gene under the control of a tapetum-specific promoter, were used as starting material. The method is based on the following principles: processing of the tissue on crushed ice, no fixation but a pretreatment with spermidine, partial dehydration with acetone, and a final embedding in a water-miscible glycol methacrylate resin at 5-degrees-C. This method was used to set up a sensitive beta-glucuronidase histochemical assay with a high resolution. A succinate dehydrogenase assay was included as a control for tissue and cell viability as well as a standard for the relative metabolic activities between different tissues and cell types.