迭代漂白扩展复用技术(IBEX)可同时识别所有主要视网膜细胞类型。

IF 3.3 3区 生物学 Q3 CELL BIOLOGY Journal of cell science Pub Date : 2024-11-14 DOI:10.1242/jcs.263407
Aanandita A Kothurkar, Gregory S Patient, Nicole C L Noel, Aleksandra M Krzywańska, Brittany J Carr, Colin J Chu, Ryan B MacDonald
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引用次数: 0

摘要

要了解组织的多细胞组成,以及它在发育、老化和/或疾病过程中是如何改变的,我们必须对完整的细胞景观进行可视化。目前,我们结合多种细胞标记物的能力有限,这阻碍了我们的工作。为了克服这一问题,我们将一种名为 "迭代漂白扩展复用(IBEX)"的高度复用免疫荧光(IF)技术应用于斑马鱼视网膜。我们优化了荧光抗体微连接技术,在单个组织上进行连续多轮标记,用 11 种细胞特异性抗体同时观察所有主要视网膜细胞类型。我们进一步调整了 IBEX,使其与荧光转基因报告基因系、原位杂交链反应(HCR)和整装免疫荧光(WMIF)兼容。我们在多个阶段应用 IBEX 研究视网膜发育过程中神经胶质细胞和神经元之间的时空关系。最后,我们在绿松石鳉(Nothobranchius furzeri)和非洲爪蛙(Xenopus laevis)身上进行了测试,从珍贵的组织中收集了大量信息,从而证明了 IBEX 跨物种的实用性。这些技术将彻底改变我们对任何生物体内多种细胞类型进行可视化的能力,而这些生物体内的抗体都是现成的。
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Iterative Bleaching Extends Multiplexity (IBEX) facilitates simultaneous identification of all major retinal cell types.

To understand the multicellular composition of tissues, and how it is altered during development, ageing and/or disease, we must visualise the complete cellular landscape. Currently, this is hindered by our limited ability to combine multiple cellular markers. To overcome this, we adapted a highly multiplexed immunofluorescence (IF) technique called Iterative Bleaching Extends Multiplexity (IBEX) to the zebrafish retina. We optimised fluorescent antibody micro-conjugation to perform sequential rounds of labelling on a single tissue to simultaneously visualise all major retinal cell types with 11 cell-specific antibodies. We further adapted IBEX to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and wholemount immunofluorescence (WMIF). We applied IBEX at multiple stages to study the spatial and temporal relationships between glia and neurons during retinal development. Finally, we demonstrate the utility of IBEX across species by testing it on the turquoise killifish (Nothobranchius furzeri) and African clawed frog (Xenopus laevis) to glean large amounts of information from precious tissues. These techniques will revolutionise our ability to visualise multiple cell types in any organism where antibodies are readily available.

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来源期刊
Journal of cell science
Journal of cell science 生物-细胞生物学
CiteScore
7.30
自引率
2.50%
发文量
393
审稿时长
1.4 months
期刊介绍: Journal of Cell Science publishes cutting-edge science, encompassing all aspects of cell biology.
期刊最新文献
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