Il 6 sc 7920

The method for protein from total kidney homogenate was extracted according to the manufacturer’s recommended protocol (Cwbiotech, Beijing, China). Proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare, Freiburg, Germany). After transfer, 5% skimmed milk powder was used to block the membranes at room temperature for 1 h. Then, the membranes were pre-incubated with primary Foxp3 (sc-53,876, 1:500 dilution), IL-6 (sc-7920, 1:500 dilution) (Santa Cruz Biotechnology, California, USA), anti-IL-10 (ab9969, 1.5 μg/mL dilution), anti-IL-17 (ab79056, 1.5 μg/mL dilution) (Abcam Ltd., Hongkong, China) and GAPDH (1:1000 dilution) (Danvers, MA, USA) antibodies at 4 °C overnight and then incubated with secondary antibodies following three washes with TBST. Then, to detection of the expression level of the protein of interest using an enhanced chemiluminescence (ECL, Millipore, Billerica, MA, USA) western blot detection kit and a chemiluminescent imaging system (Bio-Rad, USA)..

After deparaffinizing the tissues, 3% hydrogen peroxide in methanol was added and the tissues were left for 10 min to remove the endogenous peroxidase, and the antigen retrieval step was done in 0.1 M sodium citrate buffer on a hot plate. After preventing the nonspecific binding normal serum step, the primary antibody binding step was done for 1 h at 4 °C such as TNF-α (MBS175453, MY BioSource, San Diego, CA, USA), IL-6 (sc-7920, Santa Cruz, TX, USA), IL-13 (sc-1776, Santa Cruz), IL-10 (sc-73309, Santa Cruz), NF-κB (51-0500, Invitrogen, Carlsbad, CA, USA), COX-2 (ab15191, Abcam, Boston, MA, USA), and PGE₂ (bs-2639R, Bioss, Woburn, MA, USA). To detect NF-κB protein or the nucleus, immunofluorescent analysis was done with Alexa Fluor 488-conjugated secondary antibody (A3273, Invitrogen) and DAPI (62249, ThermoFisher Scientific, Waltham, MA, USA), respectively, and the results were acquired using a K1-Fluo confocal microscope (Nanoscope System, Daejeon, Republic of Korea). However, for measuring COX-2 protein or PGE₂, one immunohistochemical analysis was conducted after binding pan-specific secondary antibody, and all sectioned tissues were treated with streptavidin peroxidase (Vector Laboratories Universal Quick Kit, Burlingame, CA, USA).