Automated fluorescence microscope

We euthanized the mice 1 day after MCAO, removed the brain tissue rapidly, and sliced it into 4.0 μm sections. Then, the sections were incubated for 30 min at 37°C in dihydroethidium (DHE, Sigma–Aldrich) solution, protected from light. The sections were rinsed thrice after incubation, then we randomly selected three brain slices from the ischemic region and observed ROS‐positive cells in the penumbra with an automated fluorescence microscope (Olympus, Japan). Fluorescence intensity was quantified and averaged using ImageJ, with the sham group serving as a reference for quantification.
Mice were euthanized with an overdose of isoflurane 1 day after reperfusion. Malondialdehyde (MDA) and glutathione (GSH) levels were evaluated in the collected fresh brain tissues using commercial assay kits (EYKITS, Shanghai, China), rigorously conducted following the protocol. Statistical analysis was performed using five mice in each group.

For immunofluorescence analysis, infected Vero cells in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and then blocked for 15 min at 25 °C with a blocking solution that contained 3× rinsing with 3% BSA in PBS with fish gelatin (PBSA). Fixed cells were incubated for 2 h at 25 °C with the primary antibody (anti-HA) diluted in blocking solution. The secondary antibody (Cy3-conjugated anti-mouse IgG) was diluted in blocking solution and incubated with the cells for 1 h at 25 °C, followed by dilution with 546 phalloidin blocking solution (Invitrogen) for 40 min at 25 °C. The cells were washed five times with PBSA after each antibody treatment. Finally, cells were treated with ProLong Gold reagent with DAPI (Invitrogen). Fluorescence was analyzed microscopically with an automated fluorescence microscope (Olympus, Tokyo, Japan), and the cells were photographed with an Olympus DP70 digital camera (Tokyo, Japan).

De-paraffinization of 4-micron formalin-fixed, paraffin-embedded tissue sections was performed before conducting a pre-treatment step of heat-induced epitope retrieval (HIER) using SPoT-Light Tissue Pretreatment Solution (Thermo Fisher Scientific) at pH 7. A proteolytic digestion of tissue sections was then carried out using Protease 1 (Abbott Molecular, Des Plaines, IL, USA), followed by a rinse in saline-sodium citrate buffer (SSC). The ZytoLight SPEC ROS1 Dual Colour Break Apart Probe (ZytoVision, Bremerhaven, Germany) was used, followed by denaturation at 95 °C for 5 min and overnight hybridization at 37 °C. The slides were then dehydrated and counterstained with SlowFade Gold DAPI (Invitrogen, Waltham, MA, USA). At least 50 tumor nuclei per case were evaluated for interphase signals using an epifluorescence microscope (Zeiss, White Plains, NY, USA) and an automated fluorescence microscope (Olympus, Tokyo, Japan) equipped with cell imaging and analysis software (BioView, Rehovot, Israel). Cases were categorized as ROS1 FISH positive if they demonstrated at least 15% of cells with split signals at least one signal distance apart or an isolated centromeric 30 (green signal) pattern. This 15% cutoff was determined by in-house validation and adheres to international guidelines [15 (link)].