Immediately after imaging, rats were injected with a ketamine-xylazine cocktail and transcardially perfused with ice-cold saline and freshly prepared 4% PFA in phosphate buffer (0.1 M, pH 7.2). Brains were extracted and kept in 4% PFA overnight at 4°C, transferred to 30% sucrose at 4°C until saturated, and embedded in OCT using a dry ice and ethanol slurry. Brain tissue was cut coronally into 40 μm sections using a cryostat and separated into 6 series. Brain tissue from 9 sonicated animals and 2 control animals were analysed using immunohistochemistry; 6-10 sections were analysed per animal. Sections were stored in cryoprotectant at -20°C.

Prior to staining, brain sections were washed to remove cryoprotectant (wash buffer: 0.1% Triton X-100 in PBS; 10 min × 3 washes). For antigen retrieval, sections were treated with Rodent Decloaker (Biocare Medical; diluted 1:10 in ddH2O) at 70-80°C for 7 mins, and immersed in an ice bath for 1 h 27. Sections were washed, incubated in blocking solution for 1.5 h at room temperature (blocking solution: 0.1% Triton X-100, 5% donkey serum, 1% BSA), in primary antibodies for 48 h at 4°C, washed, incubated in secondary antibodies for 2 h at room temperature, and washed. Sections were mounted using Fluoromount-G (Invitrogen, USA).

The following anti-rat primary antibodies were used: to stain blood vessels, mouse anti-rat endothelial cell antibody 1 (RECA1; MCA970R; BioRad, Irvine, USA), mouse anti-PECAM-1 (also known as CD31, ab24590), and mouse anti-glucose transporter 1 (GLUT1, ab40084); to stain astrocytes, chicken anti-GFAP (ab4674); to stain neutrophils, rabbit anti-myeloperoxidase (MPO, ab9535). The following secondary antibodies were used: goat anti-chicken Alexa Fluor 488 (ab150169, abcam; Cambridge, USA), donkey anti-rabbit Alexa Fluor 568 (ab175692), donkey anti-mouse Alexa Fluor 647 (ab150107), goat anti-chicken Alexa Fluor 647 (ab150171). Primary antibodies for MPO and RECA-1 were optimized in spleen and liver tissue sections as positive controls. All antibody company codes that begin with 'ab' were from Abcam (Cambridge, USA).

Immunofluorescence was detected using a Nikon A1 laser scanning confocal microscope (Tokyo, Japan) or Zeiss Cell Observer spinning disk confocal microscope (Oberkochen, Germany), using a 20X air or 63X oil-immersion objective lens. Z-stacks were acquired using a step-size of 2 µm or 1 µm, respectively. Images were compiled using the Bio-Formats plugin in FIJI (Fiji Is Just ImageJ 28).

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