Fluorescent imaging of glutathione distribution in frozen tissue slices based on TLC tracing technology

Abstract

The distribution of glutathione (GSH) in liver tissue is an important indicator for disease assessment. GSH depletion increases the susceptibility to oxidative stress and is involved in many diseases. Although mass spectrometry imaging (MSI) is a powerful tool for analysing the distribution of metabolites, it is difficult to image the distribution of reactive metabolites, such as the GSH bearing thiol group. In addition, MSI needs high-end instruments and takes a long time. In this study, we aimed to construct a methodology to trace GSH in frozen tissue slices onto a thin layer chromatography (TLC) plate by labelling with a fluorescent dye in situ. When a frozen tissue slice was attached to the functional TLC plate, the thiol-specific fluorescence labelling agent for TLC imaging (tFLAT) immediately reacted with GSH in the frozen liver tissue slice to produce a high-polarity GSH adduct (tFLAT-GSH). After the separation of unreacted tFLAT on the TLC plate by expansion using a low-polarity solvent, fluorescence signals derived from tFLAT-GSH were observed in situ. We also monitored the decrease in GSH in liver tissue slices when mice were treated with acetaminophen (APAP), which causes acute liver inflammation. The present method successfully detected GSH in the mouse liver tissue slices. This study provides a rational strategy for tracing target metabolites within frozen tissue slices on TLC plates for the first time.