Ab18736

Immunolabelling with rabbit polyclonal TNX (dilution 1:200, sc‐25717, Santa Cruz Biotechnology, Santa Cruz, CA, USA), calretinin (dilution 1:500, CG1 and 6B3, Swant, Mountain View, CA, USA) and calcitonin gene‐related peptide CGRP (dilution 1:400, ab36001, Abcam, Cambridge, MA, USA; dilution 1:400; ABS026‐05‐02; Thermo Fisher, Waltham, MA, USA) was assessed in human and mouse specific stomach regions. Additionally, choline acetyl transferase (ChAT) (dilution 1:400, ab18736, Abcam) was used in brainstem sections.

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.

The D54 and U251 GBM cell lines were cultured on glass coverslips overnight in DMEM/F12 medium supplemented with FBS. Single cells from dissociated xenograft cell cultures were plated on glass coverslips pre-coated with poly-l-ornithine. The coverslips were fixed with a 4% PFA solution for 10 min at room temperature, washed with cold PBS 3 times for 5 min, and permeabilized (PBS with 0.25% Triton X-100) for 10 min. The coverslips were blocked (PBS with 10% donkey serum and 0.1% Tween-20) for 30 min. Primary antibody mouse anti-MMP9 (Abcam, ab119906, Cambridge, United Kingdom) and sheep anti-ChAT (Abcam ab18736) were diluted 1:100 in blocking solution and applied at room temperature for 1 h. The coverslips were washed 5 times for 5 min with PBST (PBS with 0.1% Tween-20). Secondary antibody Donkey anti-Mouse Alexa Fluor 555 (ThermoFisher Scientific) and DAPI diluted in blocking solution were applied at room temperature for 1 h. The coverslips were washed 5 times for 5 min with PBST and then mounted with Fluoromount G Mounting Solution (Electron Microscopy Sciences Cat. #17984-25, Hatfield, PA, USA). Images were acquired using an A1R Nikon laser scanning microscope with a Plan Apo 60×/N.A.1.40 oil objective.