Extravidin cy3

After recording, slices underwent immunofluorescent staining, in order to determine the neurochemical content of examined neurons. Slices were fixed overnight in 4% formaldehyde at 4°C. Fixed, free-floating sections were blocked and permeabilized with 10% NDS and 0.6% Triton X-100 in PBS, respectively, at 4°C overnight or for 3 h at RT. Subsequently, after washing in PBS, sections were incubated with a primary antibody solution: mouse anti-RLN3 (1:15, Supplementary Table 1), rabbit anti-proCCK (1:200, Supplementary Table 1), ExtrAvidin^®-Cy3 (1:200, Sigma-Aldrich), 2% NDS and 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 48-72 h at 4°C and, after several washing steps (in PBS), with secondary antibody solution: anti-mouse Alexa 647 (1:400) or anti-mouse Alexa 488 (1:400), anti-rabbit Alexa 647 (1:400), and 2% NDS in PBS at 4°C overnight. Slices were mounted onto glass slides, coverslipped with Fluoroshield and imaged using a fluorescence microscope (Axio Imager M2, Zeiss, with an A-Plan 10×/0.25 or EC Plan Neofluar 20×/0.25 objective). A lack of RLN3 or CCK immunoreactivity was not used as a prerequisite for assigning an NI neuron as non-RLN3 or non-CCK, due to possible dilution of antigen by the intrapipette solution during patch-clamp recording.

Standard immunofluorescence was used to determine microglia activation and vagal afferent density in the hindbrain. Sections were incubated overnight with a primary antibody against ionized calcium-binding adaptor molecule 1 (Iba-1, Wako, Richmond, VA, USA; Cat#019-19741, RRDI: AB_839504) followed by Alexa-488 secondary antibody to visualize microglia activation as previously described [5 (link)]. In addition, hindbrain sections were incubated with GSL I-isolectin B4 biotin-conjugated (IB4, Vector Laboratories Cat#B-1205, RRDI: AB_2314661) overnight followed by ExtrAvidin-CY3 (Sigma-Aldrich, St. Louis, MO, USA; Cat#E-4142) for 2 h to visualize primary unmyelinated vagal afferents innervating the GI tract as previously described [8 (link)]. Sections were mounted in ProLong (Molecular Probes, OR) and examined under a Nikon 80-I fluorescent microscope. The area fraction of Iba1 was analyzed using Nikon Elements AR software as previously described [18 (link),23 (link)].

Two weeks later, mice were given an anesthetic overdose and perfused with heparinized saline followed by 4% paraformaldehyde in 0.1M PB. The brain and spinal cord were remove and post-fixed in the same fixative at room temperature for 2h then transferred to 20% sucrose in 0.1M PB overnight at 4°C. Frozen coronal sections through the cervical enlargement (C7/8) were cut at 40 μm for tracers histochemistry processing. To visualize BDA-labeled CST fibers, free-floating sections were incubated at room temperature for 1 h in 3% donkey serum with conjugated ExtrAvidin Cy3 (1:3000; Sigma). To visualize DAF 488-labeled fibers, sections were incubated with the primary antibody (1:400 rabbit anti-alexa Fluor 488; Molecular probes) overnight at 4°C. After washing with PBS 0.1_M, sections were incubated in the secondary antibody at room temperature for 2h (1:500 donkey anti rabbit conjugated to FITC, Jackson ImmunoResearch). Sections were washed, mounted on gelatin-coated slides, air dried overnight, and cover slipped with Vectashield (Vector Laboratories).

GR-MF were seeded into 24-well culture plates at a concentration of 1 × 10⁴ cells/well, in 500 μL standard culture medium, and fixed after 2 days culture. Cells were washed with HBSS solution and fixed in 4 % paraformaldehyde in PBS buffer, pH 7.4 for 15 min. After fixation, single-cell preparations were washed several times in PBS and further treated with 50 nM NH₄Cl (30 min), 0.05 % saponin/PBS (30 min) and 0.05 % gelatin/0.05%saponin/PBS (30 min). Cells were treated with primary antibody, rat monoclonal anti-CCL11/eotaxin (R&D Systems) 5 μg/mL diluted 0.05 % gelatin/0.05%saponin/PBS, at 4 °C, overnight. Thereafter, cells were treated with a biotinylated IgG goat anti-rat antibody (Vector Labs) at the dilution indicated by the manufacturer. Fluorescence labeling was performed with extravidinCy3 (Sigma-Aldrich, Inc., St. Louis, MO) diluted 1:600. In both protocols, cells were examined with an Olympus epifluorescence microscope, and images were acquired using the ImagePro program.

Using immunocytochemistry, we also detected the presence of the telomerase enzyme in analyzed DPSCs. For this analysis, DPSCs from the 2nd passage were seeded in a concentration of 5000 cells per cm² and cultivated in chamber slides (Nalge Nunc International Corporation, Rochester, NY, USA) for two days. Before immunostaining, the adherent cells were fixed using 10% formaldehyde. After a thorough washing with phosphate-buffered saline (PBS), a 0.5% solution of Triton (250 mL Triton (Sigma-Aldrich) and 500 mL PBS) was used for 10 min to facilitate antibody penetration. Following a 20 min incubation of the sections with PBS containing a goat serum (1:20; Jackson ImmunoResearch Labs, West Grove, PA, USA), they were treated with a primary liquid mouse monoclonal antibody NCL-L-hTERT aimed against human telomerase reverse transcriptase (1:50; Novocastra, Leica Biosystems, Nußloch, Germany) for 60 min. After washing, the sections were treated with the secondary anti-mouse antibody IgG2a2b2c. For immunostaining, the samples were treated with ExtrAvidin−Cy3 (Sigma-Aldrich). We counterstained cell nuclei with 4′-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 5 min and observed samples with a BX51 Olympus microscope. Images were overlapped using Adobe^® Photoshop CC 2020 (Adobe Systems, San Jose, CA, USA).

A total of 1 × 10⁷ cells were fixed in 1 ml HMI-9 medium containing 2% formaldehyde (5 min, RT). Cells were washed three times with 1 ml PBS (1,000 × g, 5 min, RT) and settled on poly-l-lysine-coated slides. The cells were permeabilized in 0.2% Igepal CA-630/PBS (5 min, RT) and then blocked in 1% BSA/PBS (1 h, RT). After removal of the blocking solution, ExtrAvidinCy3 (Sigma) was applied at 1:100 in 0.1% BSA/PBS including 5 μg/ml Hoechst stain (30 min, RT, dark). Slides were washed with PBS and mounted with Vectashield (Vecta Laboratories Inc.). Images were captured with a Leica DMI 6000B microscope and processed with the software Fiji.