Ultra sonication

After completion of experiments, animals were sacrificed using isoflurane anesthesia (Abbott Laboratories Ltd., Queenborough, Kent, UK), and the spinal cord samples were preserved at −80 °C. The spinal cords were placed in lysis buffer and subjected to ultra-sonication (Misonix, Inc., Farmingdale, NY, USA) and centrifugation at 15,000 RPM for half an hour at 4 °C. The supernatant was quantified by the Bradford protein assay. Protein denaturation was done by heating at 90 °C for 10 min and separated by 12% SDS-PAGE. The proteins were transmitted onto a polyvinylidene fluoride membrane (Pall, Ann Arbor, MI, USA) and kept for blocking with 5% skimmed milk in tris-buffered saline (0.05% Tween 20 in tris-buffered saline). The membrane was incubated overnight with the following primary antibodies at 4 °C; anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-GLP-1R (1:200; Abcam, Cambridge, UK). After overnight incubation, the samples were washed with Tris-buffered saline with 0.1% Tween 20. Then, the samples were incubated with horseradish peroxidase-conjugated anti-rabbit (1:5000; Leadgene Biomedical, Tainan, Taiwan) for three hours at room temperature and identified using Enhanced Chemiluminescence Western Blotting Kit (Advansta, Menlo Park, CA, USA). The intensity of blots was quantified using the ImageJ software.

On day 7 after osmotic pump installation, animals were sacrificed using isoflurane anesthesia (Abbott Laboratories Ltd., Queenborough, Kent, UK), and the spinal cords were stored at −80 °C until further use. The samples were lysed by ultrasonication (Misonix, Inc. Farmingdale, NY, USA) in a lysis buffer, followed by centrifugation at 15,000 RPM for 30 min at 5 °C. The supernatant was carefully isolated and quantified. Proteins were denatured by heating at 90 °C for ten minutes and isolated by 12% SDS-PAGE. Further, they were transmitted onto a polyvinylidene fluoride membrane (PVDF) (Pall, Ann Arbor, MI, USA) and blocked with 5% skimmed milk in Tris-buffered saline (0.05% Tween 20 in Tris-buffered saline). The PVDF membrane was incubated overnight with primary antibody at 4 °C: anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-CD-11b (1:200; Abcam, Cambridge, UK). After 12 h, the membrane was washed with Tris-buffered saline with 0.1% Tween 20. Finally, the samples were incubated with horseradish peroxidase-conjugated anti-rabbit (1:5000; Leadgene Biomedical, Tainan, Taiwan) for four hours at 30 degrees and identified using Enhanced Chemi-luminescence Western Blotting Kit (Advansta, Menlo Park, CA, USA). The intensity of blots was quantified using the ImageJ software.