Leptin neutralizing antibody

Recombinant human leptin and AG490 were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). UO126 and LY294002 were purchased from Cell Signaling Technology (Boston, MA, USA). Fasudil was purchased from Abcam (Cambridge, MA, USA) and the leptin-neutralizing antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Topical inhibition of leptin signaling was conducted by perineural injection of leptin inhibitors. According to previous reports, leptin neutralizing antibody (R&D systems, Germany) has been used for perineural administration^{10 (link)}. leptin neutralizing antibody was dissolved in sterilized PBS, therefore same PBS has been used for vehicle injection as controls. Perineural injection (20 ng/mouse/day) has accompanied using the method described previously^{10 (link)}, thus drugs were injected locally to perineural region of ipsilateral sciatic nerve. The first injection was done immediately after the surgery and was continued by single daily injections for 14-days in total. Every mouse was tested 24 h after the latest injection to avoid the acute pharmacological effect of inhibitors.