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Author:
Date:
2021-04-05
[Abstract] Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can ...
[摘要] [摘要]微管（MT）是细胞骨架中最刚性的组成部分。然而，它们在细胞环境中经常显得高度弯曲，并且控制它们整体形状的机理了解甚少。当前，体外微管分析主要依靠电子显微镜进行高分辨率分析，而全内反射荧光（TIRF ）显微镜则可以对活的荧光标记的微管和相关蛋白进行成像。为了对具有微米曲率的微管进行三维分析，我们开发了一种在体外聚合MT的检测方法 用类似于常规TIRF显微镜操作规程的方式将MT种子的MT粘附到载玻片上。除去游离的荧光分子，并通过灌注固定MTs。然后可以使用带有Airyscan模块的共聚焦显微镜观察MT，以获得更高的分辨率。该协议允许对保留其原始三维形状并与高分辨率免疫荧光检测兼容的微管进行成像。
[背景]微管（MT）是通过异源二聚体的组合制成的聚合物α和β微管蛋白，并且是细胞骨架的主要成分。他们参与了细胞功能的基本机制，如有丝分裂，细胞内转运，胞质分裂和细胞形态的维持（Akhmanova和Steinmetz，2015）。尽管MT本身具有很高的刚性，但它们通常会在细胞中弯曲并产生一些蛋白，从而弯曲微管（Brangwynne等人，2006; Bechstedt等人，2014; Leung等人，2020; Cuveillier等人，2020 ...
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Author:
Date:
2020-10-05
[Abstract] Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to transdifferentiate toward α-SMA+ myofibroblasts leading to interstitial fibrosis. Even if precise pericytes identification requires transmission electron microscopy and the co-immunostaining of several markers (i.e., Gli, NG2 chondroitin sulphate proteoglycan, CD146, desmin or CD73) and emerging new markers (CD248 or TEM1, endosialin), previous studies suggested that PDGFR-β could be used as marker for renal pericytes characterization. ...
[摘要] [摘要]越来越多的证据表明，肾小管周围的毛细血管周细胞是慢性肾脏病（CKD）以及急性肾损伤（AKI）早期形成疤痕的成纤维细胞的主要来源。在败血症和I / R（缺血再灌注）损伤诱导的AKI的猪模型中，我们证明了肾周细胞能够向α- SMA +肌成纤维细胞转分化，从而导致间质纤维化。即使精确周细胞识别需要透射电子显微镜和几个标志物联合免疫（即。，的Gli ，NG2硫酸软骨素蛋白聚糖，CD146，结蛋白或CD73）和新兴的新的标志物（CD248或TEM1，唾液酸蛋白），以往的研究表明，PDGFR-β可用作肾周细胞表征的标志物。最近，对PDGFR-β和α-SMA进行了双重免疫荧光染色，以鉴定在纤维化发展的早期受损激活的周细胞（PDGFR-β + /α-SMA +细胞）。我们的数据强调了肾周细胞在败血症和I / R相关性AKI的生理病理中的关键作用。在该协议中，我们描述了猪福尔马林固定石蜡包埋（FFPE）肾脏活检中PDGFR-β和α-SMA双重免疫荧光染色的程序以及图像分析和定量方法。
[背景】肾脏纤维化被认为是主要负责肾脏疾病的进展，其与肾的损伤后的容量有限，再生有关。进行性肾脏疾病中间质纤维化的主要来源（Simone等人，2014 ; ...
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