Dihydroethidium (DHE), an oxidative fluorescent dye, was used to detect ROS in the spinal cord after SNT surgery. To avoid reduction of ROS fluorescence during immunostaining, DHE (Sigma, 10 μg in 5 μl ACSF) was injected by direct lumbar puncture between L5 and L6 vertebrae of the spine, using a 10-μL Hamilton syringe (Hamilton Bonaduz AG) with a 31G needle. Two hours after the DHE injection, the mice were anesthetized and perfused intracardially with 20 ml PBS followed by 20 ml of cold 4% paraformaldehyde (vol/vol) in PBS. The spinal cord was harvested similar to that for immunostaining and stored in dark. The samples were then cryocut within 48 h after perfusion fixation and imaged within 2 h after the slice mounting. DHE, DAPI signals, and the area of the microglial cell body were quantified with ImageJ software (US National Institutes of Health) by masking non-microglial cells. The oxidation of DHE in vivo can generate multiple fluorescent products with overlapping spectra, which may not be exclusively located in the nucleus.

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