| Non-separate Mouse Sclerochoroid/RPE/Retina Staining and Whole Mount for the Integral Observation of Subretinal Layer
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Author:
Date:
2021-01-05
[Abstract] The subretinal layer between retinal pigment epithelium (RPE) and photoreceptors is a region involved in inflammation and angiogenesis during the procession of diseases such as age-related macular degeneration. The current protocols of whole mounts (retina and RPE) are unable to address the intact view of the subretinal layer because the separation between retina and RPE is required, and each separate tissue is then stained. Non-separate Sclerochoroid/RPE/Retina whole mount staining was recently developed and reported. The method can be further combined and optimized with melanin bleaching ...
[摘要] [摘要]视网膜色素上皮（RPE）和感光细胞之间的视网膜下层是与年龄相关的黄斑变性等疾病进程中涉及炎症和血管生成的区域。由于需要在视网膜和RPE之间分离，因此整个安装（视网膜和RPE）的当前方案无法解决视网膜下层的完整视图，然后对每个单独的组织进行染色。最近开发并报道了非分离的巩膜脉络膜/ RPE /视网膜整装染色。该方法可以与黑色素漂白和组织清除进一步结合和优化。在这里，我们描述了非色素和色素小鼠硬化脉络膜的步骤/ RPE /视网膜整个安装，包括眼球准备，染色，安装和共聚焦扫描。此外，我们提出了巩膜脉络膜/ RPE /视网膜整个支架中的RPE，视网膜下小胶质细胞和邻近的感光体的共聚焦图像。
[背景]在眼睛视网膜是由视网膜色素上皮（RPE）包围，脉络膜和巩膜。通常，在整装染色中，视网膜组织与脉络膜/ RPE分开，并且分开的视网膜和脉络膜/ RPE的每个部分都被染色。因此，视网膜或脉络膜/ RPE的单独的整体染色不能解决完整的视网膜下信息。最近，开发了脉络膜/ RP E /视网膜整个安装方案（Kim等，2016； ...
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| Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo
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Author:
Date:
2013-10-20
[Abstract] Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, ...
[摘要] 双分子荧光互补（BiFC）测定是用于使用活细胞成像或固定细胞直接观察蛋白质 - 蛋白质相互作用的方法。这里描述的协议是基于我们最近的论文描述人类染色质衔接子和转录辅因子Brd4与p53肿瘤抑制蛋白的功能联系（吴等人。2013年）。 BiFC首先由Hu等人（2002）使用增强的黄色荧光蛋白（EYFP）的两种非荧光蛋白片段描述，所述荧光蛋白是Aequorea victoria GFP变体蛋白，分别融合Rel家族蛋白和bZIP家族转录因子，以调查这两个家庭成员在活细胞之间的相互作用。通过引入突变以降低其对pH和氯离子的敏感性，从而产生称为金星荧光蛋白的超增强YFP，而在37℃下不显示减少的荧光，如通常用EYFP观察到的，YFP被改进（Nagai等al。，2006）。荧光信号通过两个非荧光片段（例如，金黄N末端1-158个氨基酸残基，称为Venus-N）及其C-末端159-239个氨基酸的互补而再生残基，命名为Venus-C;参见图1A和Gully等人，2012; Ding等人，2006; ...
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