Cm1520

Mice were deeply anaesthetized with isoflurane, and perfused with a buffered 10% formaldehyde solution (Mildform 10N; FUJIFILM Wako). The spinal cords obtained from the lumbar enlargements were collected as soon as possible. The tissues were immersed in the same fixative at 4°C for 4 h or overnight, and equilibrated in 20% sucrose overnight at 4°C before cryoprotection. Sections with a thickness of 10 μm were prepared using a cryostat microtome (CM1520, Leica Microsystems), mounted on APS‐coated slide glasses (S8441; Matsunami, Osaka, Japan), and stored at –70°C until use. Sections were washed three times with 0.1 M PBS (pH 7.6) for 10 min, then treated with 2% goat serum for 60 min. The sections were primarily incubated with a goat anti‐nNOS antibody (1:100, ab1376; Abcam, Cambridge, UK) and a rabbit anti‐mGluR2 and anti‐mGluR3 antibody (1:100, ab6438; Abcam) for 2 days at 4°C. After washing, sections were secondarily incubated with a donkey anti‐goat IgG H&L conjugated with Alexa Fluor488 (to visualize nNOS; 1:1000, ab150129; Abcam), and a donkey anti‐rabbit IgG conjugated with rhodamine (to visualize mGluR2 and mGluR3; 1:2000 AP182R; EMD Millipore, Burtlington, MA, USA) overnight at 4°C. After washing, the sections were mounted using a medium containing DAPI (H‐1200; Vector Laboratories, Burlingame, CA, USA).

Seven days after UUO, the left kidney was obtained and transversally dissected. One-half of the kidney was fixed by immersion in near-freezing 2-methyl butane, covered with a cryoprotectant solution, and mounted on specimen holders. Next, the kidneys were cut into serial sections of 8 µm on a cryostat at −20 °C (CM-1520; Leica Microsystems, Nussloch, Germany) and later mounted on glass coverslips for staining. Kidney morphology was evaluated with hematoxylin and eosin (H&E) [5 (link)] and lipid accumulation with Nile red staining [18 (link)]. For Nile red staining, sections were washed with PBS pH = 7.4 and incubated for 10 min in darkness with 2.5 µg/mL Nile red dissolved in PBS with 1% acetone; Vectashield^® was used as a mounting medium. The photomicrographs were taken using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA). Nile red quantification was performed using Fiji [19 (link)] by ImageJ software (National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/index.htm, accessed on 20 July 2022) according to a previously reported protocol [18 (link)].

All mice were anesthetized by a single intraperitoneal injection of 60 mg/kg pentobarbital sodium (JW Pharm. Co., Ltd., Republic of Korea). They were transcardially rinsed with 0.1 M phosphate-buffered saline (PBS, pH 7.4) (Sigma, St. Louis, MO, USA) and fixed with 4% paraformaldehyde (Sigma, St. Louis, MO, USA) in 0.1 M PBS (pH 7.4) (Sigma, St. Louis, MO, USA). Their brains were removed and postfixed with the same fixative for 5 h, and the brain tissues were cryoprotected by infiltration with 30% sucrose (Sigma, St. Louis, MO, USA) for 8 h. The brain tissues were then frozen and transversely sectioned into 30-μm thickness in a cryostat (CM1520, Leica Microsystems, Wetzlar, Germany).

Frozen sections of dorsal skin tissues were embedded in the Optimal Cutting Temperature compound (Bright Cryo-M-Bed; Bright Instruments, Luton, United Kingdom) and were stored at −80°C for further processing. Next, 10 µm of frozen skin samples were sectioned with a cryostat (CM1520; Leica Microsystems, Wetzlar, Germany). Then, skin sections were stained with hematoxylin and eosin (H&E) following standard protocols. Sections were imaged and captured with the ProGres Microscope Camera (Jenoptik, Jena, Germany) with exposure settings of 1.5 ms, light at 7.0, and magnification at ×10. Epidermal thickness (µm) was quantified with ImageJ software (v.1.32; National Institutes of Health, Bethesda, MD, USA) and was measured between the epidermal surface and the epidermal–dermal junction, excluding the rete ridges. The average thickness was determined over an average of 6–10 sections/animal. For each skin section, we measured the whole length of the skin where possible (hence, at ×10 magnification, they were determined by ≥3–4 images). For each image, we took 8 points distributed evenly over the epidermis and averaged them. Hence, the data presented here is the value of average epidermal thickness/percentage of area coverage.