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。

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.