9 February 2018 Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples
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Resin embedding is widely used and facilitates microscopic imaging of biological tissues. In contrast, quenching of fluorescence during embedding process hinders the application of resin embedding for imaging of fluorescence-labeled samples. For samples expressing fluorescent proteins, it has been demonstrated that the weakened fluorescence could be recovered by reactivating the fluorophore with alkaline buffer. We extended this idea to immunofluorescence-labeling technology. We showed that the fluorescence of pH-sensitive fluorescein isothiocyanate (FITC) was quenched after resin embedding but reactivated after treating by alkaline buffer. We observed 138.5% fluorescence preservation ratio of reactivated state, sixfold compared with the quenched state in embedding resin, which indicated its application for fluorescence imaging of high signal-to-background ratio. Furthermore, we analyzed the chemical reactivation mechanism of FITC fluorophore. This work would show a way for high-resolution imaging of immunofluorescence-labeled samples embedded in resin.
© The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
Longhui Li, Longhui Li, Gong Rao, Gong Rao, Xiaohua Lv, Xiaohua Lv, Ruixi Chen, Ruixi Chen, Xiaofeng Cheng, Xiaofeng Cheng, Xiaojun Wang, Xiaojun Wang, Shaoqun Zeng, Shaoqun Zeng, Xiuli Liu, Xiuli Liu, } "Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples," Journal of Biomedical Optics 23(2), 020501 (9 February 2018). https://doi.org/10.1117/1.JBO.23.2.020501 . Submission: Received: 27 October 2017; Accepted: 12 January 2018
Received: 27 October 2017; Accepted: 12 January 2018; Published: 9 February 2018

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