Intracellular fixation permeabilization buffer plus brefeldin a kit

Macrophages in kidney were labeled and identified by flow cytometry. Briefly, kidney was harvested and minced by scissors and strainer. Acquired tissues were then fully digested using collagenase D (0.05%; Sigma–Aldrich). After filtering impurities, cells were counted and stained with allophycocyanin (APC)-CD11b (17-0112; eBioscience, San Diego, CA, USA) at 4°C for 30 min. Then they were fixed and permeabilized using Intracellular Fixation & Permeabilization Buffer (plus Brefeldin A) kit (88-8823; eBioscience) following manufacturer's protocols. After that, cells were stained with Alexa Fluor 488-NOS2 (iNOS; 53-5920; eBioscience) at 4°C for 30 min. All staining procedures were carried out in the dark. Flow cytometry analysis was performed by using a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA).