Hm430

The DRGs, spinal cord, and sympathetic ganglia were cut at a thickness of 25 μm using a freezing microtome (Leica HM430, Wetzlar, Germany), and all consecutive sections were collected. Local ST25 tissue was cut into 20-μm thick sections (Wang et al., 2018 (link); Zhang et al., 2021 (link)). All sections were mounted on slides, sealed with a drop of an anti-fluorescence quenching sealer (Abcam, Cambridge, MA, USA), and observed under laser confocal (ZEISS LSM880 + Fast Airyscan, Germany) and inverted fluorescence microscope (ZEISS Axio Scope AI, Germany) microscopes.

Mice were perfused with PBS, followed by 4% PFA in PBS. Brains were post-fixed, dehydrated and embedded in paraffin wax to prepare paraffin sections. Sections of 5-μm thickness were cut using an HM430 sliding microtome (Leica).

We utilized dihydroethidium (DHE), an ROS probe that can be delivered in vivo, to measure ROS accumulation in the ischemic penumbra. According to the protocol described in our previous study [35 (link)], a solution of 10 mg/kg DHE (Invitrogen) in 50 µL normal saline was injected via the jugular vein into mice (n = 3 per group) just before the reperfusion procedure. After 2 h following MCAO/R, the brain hemisphere was collected, immersed in 30% sucrose solution, embedded in an OCT compound, and frozen using dry ice. Ischemic-penumbra-bearing sections (50 µm thick) were obtained from each mouse using a cryostat microtome (HM430, Leica) set at −21 °C. Two sections were randomly selected from each mouse and counterstained with Hoechst33258 for 5 min at 24 °C, and then mounted with a mounting medium (FluorSave^TM, Merck-Millipore). The resulting DHE fluorescence was visualized using an LSM-700 laser scanning confocal microscope. The number of DHE-positive cells (red fluorescence) was averaged per group and expressed as a percentage of total cells (blue fluorescence), following image analysis.