Dcfda fluorescent dye

The levels of ROS in the intestinal mucosa were analyzed by 10 mM DCFDA fluorescent dye (Sigma), which was added into the suspension of intestinal mucosa for cultivation. DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2ʹ,7ʹ-dichlorofluorescein (DCF). DCF is detected by fluorescence spectroscopy with excitation and emission spectra of 495 nm and 529 nm, respectively [25 (link)].

The brains of transgenic or control flies were freshly dissected out in PBS buffer for analysis of the intracellular ROS level by incubating with 10 μM DCF-DA fluorescent dye (Sigma) for 5 min at RT. The fluorescent signals were observed using a Leica TCS SP5 II confocal microscope with 20× objective at 490 nm excitation and 525 nm emission wavelengths. The whole-brain fluorescent signals were determined using ImageJ for quantification of the ROS level. Representative images were processed using Adobe Photoshop. To determine the brain ROS activities, gstD-GFP reporter expressions in transgenic or control flies were analyzed by western blotting of total brain lysates using an anti-GFP antibody. To determine whether the gstD-GFP fluorescent reporter expression was localized to neurons, double immunofluorescence staining of brains with antibodies against GFP and ELAV was performed and observed by confocal microscope with 63× oil objective.

The levels of ROS in the intestinal mucosa were analyzed by 10 mM DCFDA fluorescent dye (Sigma), which was added into the suspension of intestinal mucosa for cultivation. DCFDA is deacetylated by cellular esterases to a non-fluorescent compound that is later oxidized by ROS into 2ʹ7ʹ–dichlorofluorescein (DCF). DCF is identified by fluorescence spectroscopy with excitation and emission spectra of 495 nm and 529 nm, respectively [16 (link)].