Ab1376

Adult male C57BL/6 mice were euthanised by the administration of a lethal dose of anaesthesia and their duodenal tissues were fixed with a 4% formalin solution (Sigma-Aldrich) for 16 hours and maintained at 4°C in 70% ethanol until they were embedded in paraffin. Six-micron-thick sections were prepared. After a citrate pretreatment, sections were incubated with goat anti-nNOS (1/100, ab1376, Abcam), sheep anti-ChAT (Choline Acetyl Transferase, 1/100, ab18736, Abcam), rabbit anti-MOR (1/100, ab10275, Abcam) or rabbit anti-Alox12 (1/100, bs3874R, Bioss) primary antibodies for 16 hours at 4°C. After washes with 1X phosphate-buffered saline (PBS), sections were incubated with fluorescein isothiocyanate (FITC)-conjugated species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA) to detect MOR or Alox12 labelling and with tetramethylrhodamine (TRITC) secondary antibodies (Jackson Laboratories) to detect ChAT labelling in dual labelling experiments. For MOR and nNOS dual labelling and Alox12 and nNOS dual labelling, sections were incubated with FITC-conjugated secondary antibodies to detect nNOS labelling and with TRITC-conjugated secondary antibodies to detect MOR/Alox12 one. Nuclei (blue) were counterstained with Hoechst 33 258. High-quality fluorescence images were captured with a Leica DM5500B microscope using a 100× oil objective.

Immunolabelling with rabbit polyclonal TNX (1:200, Santa Cruz, sc‐25717, Santa Cruz, CA, USA), calretinin (1:500, Swant, CG1 and 6B3, Marly, Switzerland), calcitonin gene‐related peptide (CGRP) (1:400, Abcam, ab36001, Cambridge, MA, USA; and Thermo Fisher, ABS026‐05‐02, Waltham, MA, USA), choline acetyltransferase (ChAT) (1:400, Millipore, AB144P, , Billerica, MA, USA), neuronal nitric oxide synthase (NOS) (1:400, Abcam, ab1376), protein gene product 9.5 (PGP9.5) (1:500, Agilent/DAKO‐Z5116, , Santa Clara, CA, USA) was assessed in human‐ and mouse‐specific GI regions.

The sections were acquired similarly as in the H&E staining procedures. After dewaxing and hydration, the slides were exposed to citrate buffer. The slides were then pretreated at 80°C and incubated with 3% H₂O₂ to block endogenous peroxide. Thereafter, the slides were treated with anti-cystathionine β-synthase (CBS, ab140600, Abcam) antibody, anti-cystathionine γ-lyase (CSE, ab151769, Abcam) antibody, or antineuronal nitric oxide synthase (nNOS, ab1376, Abcam) antibody for 2 h at room temperature. After incubation with the relevant secondary antibody, the slides were mounted and analyzed using a light microscope (Nikon).

Cells in 24-well plate were washed with PBS and fixed with paraformaldehyde 4% 10 min at RT and washed 3 times in PBS. Fixed cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), washed, and blocked in PBS/5% bovine serum albumin (BSA) for 40 min at RT. Cells were then incubated in PBS/1% BSA/0.1% saponin with a goat polyclonal anti-nNOS antibody (Ab1376, Abcam, 1:500), overnight at RT; washed in PBS/1%BSA/0.1% saponin; and incubated for 1 h with secondary antibody: Donkey anti-goat (Alexafluor 594 conjugate, Life Technologies, 1:500) and with DAPI (1:5000, Sigma). Fixed cells were then thoroughly washed in PBS/1% BSA/0.1% saponin and then in PBS and mounted in Fluoromount (Southern Biotech). Images were acquired with Leica DM2500 confocal microscope using × 63 objective.

LMMP tissues were fixed in Zamboni's fixative (4% paraformaldehyde and 0.2% piciric acid in 0.1 M PBS; #1459, Newcomer Supply, Middleton, WI) overnight at 4°C. The next day, tissues were rinsed in PBS until clear. Tissues were transferred to glass slides and incubated in blocking buffer [10% normal donkey serum (NDS), 0.5% Triton-X 100 in PBS] for 2 h at room temperature. Tissues were then incubated in appropriate primary antisera overnight at 4°C against green fluorescent protein (GFP; 1:500; ab13970, Abcam, Cambridge, MA), HuD (1:25; A-21271, Life Technologies, Eugene, OR), S100 Protein Ab-2 (S100; 1:200; RB-044-A0, Thermo Scientific, UK), choline acetyltransferase (ChAT; 1:50; AB144P, Millipore, Temecula, CA); vasoactive intestinal peptide (VIP; 1:200; Abcam), neuronal nitric oxide synthase (nNOS; 1:200; ab1376, Abcam), calretinin (1:200; CG1, Swant, Switzerland), or calbindin D-28K (1:200; AB1778, Millipore). The next day, tissues were rinsed 4 times for 10 min in PBS, then incubated in appropriate Alexa Fluor secondary antibodies at 1:200 for 2 h at room temperature. Antibody dilutent was 3% NDS, 0.5% Triton-X 100 in PBS. Tissues were then rinsed and slides were coverslipped with 2.5% PVA/DABCO. Fluorescent images were captured on an Olympus FV 100 Spectral Confocal system (Melville, NY) in the Ohio State University Campus Microscopy and Imaging Facility.

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).

The mice in all 4 groups were sacrificed immediately after completing the LPP measurements, and the urethras were harvested. The middle one-third portions of the urethras were examined with immunofluorescence staining. Each tissue sample was embedded in Tissue-Tek Optimal Cutting Temperature (OCT) Compound (Torrance, CA, USA) and then frozen, and 8-μm cryostat sections were cut and used for immunofluorescence staining of nNOS, iNOS, ERα, and ERβ. For immunofluorescence staining, the sections were permeabilized with 0.05% Triton X-100 for 5 min and blocked with 5% normal bovine serum albumin in PBS for 1 h at room temperature [26 , 27 ]. The sections were incubated with a primary antibody (goat polyclonal to nNOS, 1 : 300 dilution, Abcam (ab1376), Cambridge, UK; rabbit polyclonal to iNOS, 1 : 50 dilution, Abcam (ab3523); mouse monoclonal to ERα, 1 : 300 dilution, Abcam (ab2746); or rabbit polyclonal to ERβ, 1 : 180 dilution, Abcam (ab3577)) overnight at 4°C. The sections were washed 3 times with PBS, incubated with fluorescein isothiocyanate-conjugated secondary antibody (donkey anti-goat IgG conjugate, 1 : 200 dilution, Invitrogen (A11055), USA; goat anti-rabbit IgG conjugate, 1 : 150 dilution, ZYMED (81-6111); or goat anti-mouse IgG conjugate, 1 : 300 dilution, ZYMED (81-6511)) for 1 h at room temperature, and viewed under fluorescence microscopy.