Eukitt medium

Serial paraffin sections (3 μm thick) were obtained with a PFM slide microtome (Bio‐Optica), dried and stored until use. In brief, after dewaxing, paraffin sections were reacted with H₂O₂ (3% in deionised H₂O; 5 min) to block endogenous peroxidase, rinsed twice in phosphate‐buffered saline (PBS) and treated with normal serum blocking solution (2% in PBS; 20 min). Next, they were incubated with the polyclonal goat anti‐BChE antibody (Table 1a). After a thorough rinse in PBS, sections were incubated in a 1:200 v/v biotinylated secondary antibody solution (in PBS; 30 min; anti‐goat IgG biotinylated, BA‐5000, Invitrogen, Thermo Fisher Scientific), rinsed in PBS and incubated in avidin‐biotin‐peroxidase complex (Vectastain Elite ABC Kit, Peroxidase, Vector Laboratories), washed several times in PBS and finally incubated in 3,3‐diaminobenzidine tetrahydrochloride (Impact DAB Substrate kit, Peroxidase, Vector Laboratories, 5 min). Sections were finally counterstained with haematoxylin, dehydrated, cleared with xylene and mounted in Eukitt medium (Sigma–Aldrich). Staining was never observed when the primary antibody was omitted.

Human bioptic tissue specimens were obtained from IRCCS San Raffaele Interinstitutional Multidisciplinary BioBank (BioBIM) (Rome, Italy) and Istituto Nazionale Tumori Regina Elena (National Cancer Institute, Rome, Italy) or purchased as tissue microarrays (Biochain, Newark, CA, USA; Super Bio Chips, Seoul, Korea; Cybrdi Inc, Rockville, MD, USA). Informed consent was obtained from subjects enrolled in this study; all investigations were performed in accordance with ethical principles embodied in the Declaration of Helsinki.
Formalin-fixed paraffin-embedded human tissue sections and microarrays were deparaffinized, rehydrated in descending graded ethanol solutions and treated with 0.6% hydrogen peroxide in methanol. Heat-induced antigen retrieval was achieved in Citrate buffer, pH 6.0 (Novus Biologicals, UK). Slides were pre-incubated with Protein Block reagent (Abcam, UK) for 1 hour at room temperature, and subsequently incubated overnight at 4°C with chA1-L1 mAb (8 μg/ml) in 1% BSA/PBS. Sections were stained using the Mouse Specific HRP/DAB (ABC) kit (Abcam), counterstained with Hematoxylin (Sigma-Aldrich), dehydrated in ethanol, mounted in Eukitt medium (Sigma-Aldrich) and visualized under a light microscope (Leica Microsystems) equipped with a cooled camera (SPOT RT3, SPOT Imaging Solutions) and the IAS 2000 software for image capture.

Ten minutes before FUS, one mouse was injected intravenously (through retro-orbital route) with STE (300 µL) and one mouse was untreated. Twenty-four hours after hemispheric BBB disruption (as described above), mice were sacrificed, perfused with saline through the left ventricle and fixed with 4% PFA solution. Brains were carefully collected, fixed and conserved in formalin solution before paraffin-embedding. Four µm thick paraffin sections were then processed manually. Slides were deparaffinized in three successive baths of xylene (Hydroclear) for 10 min and then rehydrated with ethanol (100, 80, 70%). After rinsing in tap water for 10 min, slides were stained with Mayer’s Hematoxylin for 1 min. After washing, slides were stained with eosin for 15 s and rapidly rinsed in tap water. Slides were dehydrated using three baths of 100% ethanol followed by three baths in xylene and mounted with Eukitt medium (Sigma-Aldrich, St. Quentin Fallavier, France).

For histological analysis, rings were fixed in 4% formaldehyde, paraffin embedded and cut into 5 µm-slices. After von Kossa staining (silver nitrate plus nuclear fast red), slices were photographed (TM300 NIKON microscope with a digital imaging system DXM1200 NIKON). Positive staining area was quantified in the medial layer and expressed as a percentage of the total medial area. The cross-sectional area (CSA, in mm²) of the rings was measured as the total surface area of the vessel on the Von Kossa slides.
For fibrosis determination, rings were stained with 0.1% picrosirius red and mounted in Eukitt medium (Sigma-Aldrich). Quantification in the medial layer stained was made in at least ten given fields per ring and expressed as a percentage of the total medial area.
Apoptotic nuclei in aortic wall of VDN rats were identified in thoracic aorta by TUNEL staining as previously described^{19 (link)}.
All analyses were performed using image analysis software (ImageJ, National Institutes of Health, Bethesda, Maryland).

We evaluated the estrous cycle immediately after the behavioral and exercise experiments, through 4–5 consecutive vaginal lavages (with 40–50 μL of distillated H₂O) then mounted on gelatinized slides (76 × 26 mm). These procedures lasted no more than 3–5 minutes, and there were no major temporal delays between behavioral experiments and fluid collection for vaginal cytology.
The vaginal smear were desiccated at room temperature and covered with 0.1% crystal violet for 1 min, then twice washed with 1 mL H₂O and desiccated at room temperature. The slides were mounted with Eukitt medium (Sigma-Aldrich) and evaluated under an optical microscope at 1x, 5x and 20x (Zeiss Axio Imager 2). The characterization of the estrous cycle was performed according to literature^{20 (link),42} . Females were categorized for initial (metestrus) or late (diestrus) follicular phase, ovulation (proestrus), or luteal phase (estrus)^{20 (link),42} .