ELISA for TNF-α, IL-1β and IL-6 in paw inflammatory tissue

Samples were pulverized in liquid nitrogen, resolved in cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing protease inhibitor cocktail (10% v/v; Bio Basic Inc., Markham, ON, Canada), sonicated on ice (5×5 sec), stored for 1 h at 4°C, and then centrifuged at 13,201 × g for 30 min at 4°C to obtain the protein extract. TNF-α, IL-1β and IL-6 were determined using a Rat TNF-α ELISA Kit (cat. no. ELR-TNFalpha-001), Rat IL-1β ELISA Kit (cat. no. ELR-IL1beta-001) and Rat IL-6 ELISA Kit (cat. no. ELR-IL6-001; all Raybiotech Corp., Atlanta, GA, USA), respectively.