Mouse anti inos

In this study, evodiamine (purity >98%), dimethylsulfoxide (DMSO), and type II collagenase were obtained from Solarbio (Beijing, China). Mouse IL-1β was purchased from Novoprotein (Suzhou, China). The following primary antibodies were utilized in the present study: mouse anti-collagen II, mouse anti-ADAMTS-5, mouse anti-COX-2, mouse anti-IκB, and mouse anti-GAPDH were purchased from Affinity Biosciences (Beijing, China). Mouse anti-iNOS, mouse anti-MMP-13, and mouse anti-Laminb1 were acquired from Proteintech (Wuhan, China). Primary antibodies to mouse anti-p65 were obtained from Cell Signaling Technology (Boston, MA, United States). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM)/F12 were acquired from Gibco (Grand Island, United States). And all ELISA kits were acquired from Cusabio (Wuhan, China).

Microglial cells after 48 h exposure to TNF-α with or without inhibitors were fixed in 4% PFA-methanol free solution (ThermoFisher, Milan, Italy), and permeabilized with 0.05% Triton X-100 (Bio-Rad Laboratories, Milan, Italy) for 3–5 min at room temperature. The cells were then rinsed with DPBS, and incubated with the primary antibodies overnight at 4 °C. The primary antibodies used were mouse ant-Iba1 (1:1000, #66,827–1-Ig, Proteintech, DBA, Milan, Italy), rabbit anti-TMEM119 (1:1000, #66,948–1-Ig Proteintech, DBA, Milan, Italy), mouse anti-iNOS (1:1000, #18,985–1-AP Proteintech, DBA, Milan, Italy), and rabbit anti-TNF-α (1:1000, #3707; Cell Signaling Technology, DBA, Milan, Italy). Secondary antibodies conjugated to Alexa 488 (1:250, #A-11029, Invitrogen, Milan, Italy) or 594 (1:250, #R37117, Invitrogen, Milan, Italy) fluorochromes were used to detect the primary antibodies. DAPI (Fluoroshield Mounting Medium with DAPI, #Ab104139 Abcam, Cambridge, UK) was used to visualize the nuclei and count cells.