Characterizing Subcutaneous Perivascular Spaces

For the characterization of gross morphology, the identified scPVS was isolated from the abdominal hypodermis region using a microscissor (blade 5 mm) and transferred into a drop of Hank's balanced salt solution (Sigma, St. Louis, MO, USA) on a slide glass. After the sample was completely air-dried at room temperature for 1–3 min, the slide glass was immersed 10 times in and taken out of dye Solution 1 for 10 s (~1/s). Additional staining with Solutions 2 and 3 was repeated using the same process. The sample was then placed in a drop of phosphate buffer solution (pH 7.2) for 20 s, washed by shaking it 10 times in distilled water for 20 s, air-dried for 3–5 min, and finally mounted with Canada balsam (Sigma). The PVS slice preparation was performed according to the previous method [6 (link)]. To confirm the DNA and RNA contents of the scPVS, the sample was kept immersed in a 0.1% acridine orange solution for 15 min, and a digital photograph was then taken using a confocal laser-scanning microscope at the wavelength of the acridine orange [19 (link)]. To verify the MCs in the scPVS, the sample was immersed in a 1% toluidine blue solution for 3 min [20 (link)].