| A Blood-retina Barrier Permeability Assay in Young Mice Using Sulfo-NHS-LC-biotin Perfusion
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Author:
Date:
2018-10-20
[Abstract] Brain and retinal vasculatures exhibit restricted vascular permeability known as blood-brain barrier and blood-retina barrier. Vascular permeability can be evaluated by perfusion of the amine reactive ester derivatives of biotin such as sulfo-NHS-LC-biotin. This protocol describes experimental procedures of sulfo-NHS-LC-biotin perfusion to evaluate retinal vascular permeability. Perfused sulfo-NHS-LC-biotin remained within vessels in wild-type postnatal day 15 (P15) retinas, confirming an intact blood-retina barrier. In contrast, sulfo-NHS-LC-biotin was occasionally detected in extravascular ...
[摘要] 脑和视网膜脉管系统表现出受限的血管通透性，称为血脑屏障和血 - 视网膜屏障。 血管通透性可以通过灌注生物素的胺反应性酯衍生物如磺基-NHS-LC-生物素来评估。 该方案描述了磺基-NHS-LC-生物素灌注的实验程序，以评估视网膜血管通透性。 灌注的磺基-NHS-LC-生物素保留在野生型出生后第15天（P15）视网膜的血管内，证实了完整的血 - 视网膜屏障。 相比之下，在灌注的 Eogt - / - >视网膜中，偶尔会在血管外空间检测到磺基-NHS-LC-生物素，这表明在没有的情况下血管完整性部分受损。 Eogt >（Sawaguchi et al。>，2017）。
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| Isolation of Murine Brain and Lung Microvascular Endothelial Cells
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Author:
Date:
2018-08-20
[Abstract] This protocol describes how to isolate murine endothelial cells from newborn mice brain and 3-month-old mice lung by modifying the original protocols (Sobczak et al., 2010; Ruck et al., 2014). We have used the protocol to analyze mRNA expression level in brain endothelial cells (Sawaguchi et al., 2017). Isolated lung endothelial cells were expanded in vitro for various downstream experiments such as gene expression analysis and cell-based signaling assay.
[摘要] 该协议描述了如何通过修改原始方案从新生小鼠脑和3个月大的小鼠肺中分离鼠内皮细胞（Sobczak et al。，2010; Ruck et al。，2014）。 我们使用该方案分析脑内皮细胞中的mRNA表达水平（Sawaguchi et al。，2017）。 分离的肺内皮细胞在体外扩增用于各种下游实验，例如基因表达分析和基于细胞的信号传导测定。
【背景】 该方案描述了从小鼠脑和肺中分离鼠内皮细胞的实验程序。 我们使用新生小鼠使用抗CD31抗体分离脑内皮细胞。 使用该方案，可以从脑内皮细胞中分离出适合于基因表达分析的2μg总RNA，尽管不能完全消除周细胞和星形胶质细胞。 我们还使用3个月大的小鼠肺使用抗CD31和抗CD102抗体分离肺内皮细胞。 将分离的肺内皮细胞在体外6cm培养皿中扩增，并用于各种下游实验，例如基因表达分析和基于细胞的信号传导测定。
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| Preparation of Knockdown Transformants of Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex
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Author:
Date:
2016-05-20
[Abstract] To prepare the knockdown transformants of the Closterium peracerosum-strigosum-littorale (C. psl.) complex, particle bombardment was applied with a newly constructed vector (pSA0104) with an endogenous constitutive promoter fused to a DNA fragment corresponding to an antisense strand of a target gene. Using a hygromycin resistance gene (aph7”), hygromycin-resistant colonies were selected. After the second screening, integration of the vector into the genome was checked by PCR and the knockdown effect was evaluated by Western blotting using a specific antibody ...
[摘要] 为了制备鬼臼藓藻（Clonsterium peracerosum-strigosum-littorale）复合物的敲低转化体，用新构建的载体（pSA0104）应用粒子轰击，所述载体具有内源性 组成型启动子，其与对应于靶基因的反义链的DNA片段融合。 使用潮霉素抗性基因（ aph7"）选择潮霉素抗性菌落。 在第二次筛选后，通过PCR检查载体到基因组中的整合，并使用针对靶蛋白的特异性抗体通过蛋白质印迹评估敲低效应。
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