Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

• Published in: Nature communications Vol. 5; no. 1; p. 3992
• Main Authors: Xiong, Hanqing, Zhou, Zhenqiao, Zhu, Mingqiang, Lv, Xiaohua, Li, Anan, Li, Shiwei, Li, Longhui, Yang, Tao, Wang, Siming, Yang, Zhongqin, Xu, Tonghui, Luo, Qingming, Gong, Hui, Zeng, Shaoqun
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 02.06.2014; Nature Publishing Group; Nature Pub. Group
• Subjects: 14/35; 14/63; 14/69; 631/1647/1888/2249; 631/1647/245; 631/378; 64/60; Animals; Bacterial Proteins; Buffers; Green Fluorescent Proteins; Histological Techniques - methods; Humanities and Social Sciences; Labeling; Luminescent Agents; Luminescent Proteins; Mice; Microscopy; Microscopy, Fluorescence - methods; multidisciplinary; Phenols; Photonics; Polymerization; Proteins; Resins; Science; Science (multidisciplinary); China
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms4992

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding. Resin embedding of specimens is widely used for microscopy but can cause the loss of fluorescence from green-fluorescent protein. Here, the authors show that these proteins can be reactivated in resin-embedded samples through the use of alkaline buffer.