Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids.
Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids.
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DOI：
10.3791/56662
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发表时间：
2017-12-13
期刊：
影响因子：
--
通讯作者：
Mogensen MM
中科院分区：
文献类型：
--
作者：
Goldspink DA;Matthews ZJ;Lund EK;Wileman T;Mogensen MM
模拟体内组织结构和形态发生的3D体外类器官的出现大大提高了研究细胞和发育生物学中关键生物学问题的能力。此外，类器官以及基因编辑和病毒基因递送方面的最新技术进展有望推动用于治疗疾病的新药的医学研究和开发。在基底基质中体外生长的类器官为研究各种蛋白质的行为和功能提供了强大的模型系统，并且非常适合荧光标记蛋白质的实时成像。然而，建立内源性蛋白在离体组织和体外类器官中的表达和定位对于验证标记蛋白的行为是重要的。为此，我们已经开发和修改了组织分离，固定和免疫标记协议的微管，中心体和相关蛋白在离体肠组织和体外肠类器官的本地化。固定剂的目的是保留类器官/组织的3D结构，同时保留抗体抗原性，并使固定剂和抗体能够良好渗透和清除。暴露于冷解聚所有，但稳定的微管，这是一个关键因素时，修改各种协议。我们发现，将乙二胺四乙酸（EDTA）浓度从3 mM增加到30 mM可有效分离小肠中的绒毛和隐窝，而3 mM EDTA足以分离结肠隐窝。所开发的甲醛/甲醇固定方案提供了非常好的结构保护，同时还保留了有效标记微管、肌动蛋白和末端结合（EB）蛋白的抗原性。它也适用于中心体蛋白ninein，尽管甲醇方案的工作更一致。我们进一步确定，微管和相关蛋白的固定和免疫标记可以用从基底基质中分离或保留在基底基质中的类器官来实现。
The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.
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有条件和可逆基因敲除一步的一代。
影响因子：
48
作者：
Andersson-Rolf A;Mustata RC;Merenda A;Kim J;Perera S;Grego T;Andrews K;Tremble K;Silva JC;Fink J;Skarnes WC;Koo BK
通讯作者：
Koo BK
影响因子：
1.2
作者：
Yoneda, Mitsuhiro;Molinolo, Alfredo A.;Goodlad, Robert A.
通讯作者：
Goodlad, Robert A.
DOI：
10.1083/jcb.109.6.2817
发表时间：
1989-12
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bacallao R;Antony C;Dotti C;Karsenti E;Stelzer EH;Simons K
通讯作者：
Simons K
影响因子：
5.3
作者：
Coquelle, FM;Caspi, M;Reiner, O
通讯作者：
Reiner, O
影响因子：
7.8
作者：
Mogensen, MM;Tucker, JB;Näthke, IS
通讯作者：
Näthke, IS