首页|二抗物种特异性对双色STORM成像的影响(特邀)

二抗物种特异性对双色STORM成像的影响(特邀)

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随机光学重建显微术(STORM)基于免疫荧光标记技术,具有原理易懂、光路简单、分辨率极高等特点,一直受到科研工作者的青睐,但分辨率的提升对抗体的特异性提出了更高的要求.相较一抗直接标记,"一抗+二抗"的间接标记法在实际应用中普适性更强.二抗相对一抗存在物种特异性的问题,生产时需要对其进行预吸附来提升物种特异性.为了探究二抗物种特异性对双色STORM成像的影响,基于经典的红细胞骨架模型中血影蛋白N端和C端的互斥位置关系,对二者使用高、低吸附二抗标记后分别进行双色STORM成像,对照模拟中有无信号串扰条件下的互相关分析结果,结果表明低吸附二抗会造成二者共定位的假象.进一步,分别通过高、低吸附二抗对MDA-MB-231乳腺癌细胞CD47和PD-L1两种膜蛋白进行双色STORM成像,结果揭示两种蛋白无共定位关系.本研究为二抗物种特异性的评估提供了一种基于红细胞骨架结构模型的超分辨成像新策略,助力双色STORM成像精准阐明蛋白分子互作关系.
Effect of Species Specificity of the Secondary Antibody on Two-Color STORM Imaging(Invited)
Stochastic optical reconstruction microscopy(STORM),a super-resolution imaging technology based on immunofluorescence,has gained popularity owing to its straightforward principle,simple optical path,and excellent spatial resolution.However,the enhanced resolution demands greater specificity from antibodies.While direct labeling with primary antibodies is an option,indirect labeling using a"primary+secondary antibody"combination is more commonly employed in practical applications.Considering the issue of species specificity with secondary antibodies,preadsorption is required to increase specificity during production.In this study,we aim to explore the effect of secondary antibody species specificity on dual-color STORM imaging.In the classical erythrocyte skeleton model,based on the mutually exclusive localization between the N-terminal and C-terminal of β-spectrin,we performed two-color STORM imaging of them by labeling them with low-and high-adsorption secondary antibodies,respectively.A comparison with cross-correlation data from simulations revealed that low-adsorption secondary antibodies led to colocalization artifacts in the STORM results.Furthermore,when employing high-and low-adsorption secondary antibodies,two-color STORM results of CD47 and PD-L1 on the MDA-MB-231 cell membrane indicated no colocalization relation between the two proteins.Therefore,this study provides an innovative super-resolution imaging strategy for evaluating secondary antibody species specificity using an erythrocyte skeleton structure model.Additionally,this approach paves the way for accurate identification of biomolecular interactions through dual-color STORM imaging.

super-resolution imagingstochastic optical reconstruction microscopyimmunofluorescenceerythrocyte membrane skeletoncross-correlation analysis

邓师禹、胡芬、侯梦迪、杨建宇、李任植、潘雷霆

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弱光非线性光子学教育部重点实验室,南开大学物理科学学院,泰达应用物理研究院,天津 300071

药物化学生物学全国重点实验室,南开大学生命科学学院,细胞应答交叉科学中心,天津 300071

南开大学深圳研究院,广东深圳 518083

极端光学协同创新中心,山西大学,山西太原 030006

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超分辨成像 随机光学重建显微术 免疫荧光 红细胞膜骨架 互相关分析

国家重点研发计划国家自然科学基金国家自然科学基金广东省基础与应用基础研究重大项目中央高校基本科研业务费专项南开大学项目中央高校基本科研业务费专项南开大学项目高等学校学科创新引智计划(111计划)

2022YFC340060032227802121742082020B030103000921220213372122021405B23045

2024

10.3788/LOP232601

激光与光电子学进展
中国科学院上海光学精密机械研究所

激光与光电子学进展

CSTPCD北大核心
影响因子：1.153
ISSN：1006-4125
年,卷(期)：2024.61(6)
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