Abstract
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.
Highlights
Longhui Li Gong Rao Xiaohua Lv Ruixi Chen Xiaofeng Cheng Xiaojun Wang Shaoqun Zeng Xiuli Liu
We showed that the fluorescence of pH-sensitive fluorescein isothiocyanate (FITC) was quenched after resin embedding but reactivated after treating by alkaline buffer
The slices were incubated with secondary antibody (FITC goat antimouse polyclonal antibody, Jackson) dilutions in PBS/0.2% Triton X100 at 37°C with gentle shaking on an oscillator for 2 h, and rinsed in 0.01-M PBS 5 min five times
Summary
Longhui Li Gong Rao Xiaohua Lv Ruixi Chen Xiaofeng Cheng Xiaojun Wang Shaoqun Zeng Xiuli Liu. We showed that the fluorescence of pH-sensitive fluorescein isothiocyanate (FITC) was quenched after resin embedding but reactivated after treating by alkaline buffer.
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