Goat anti rabbit igg h l

Azithromycin and N-acetylcysteine (NAC) were obtained from National Institutes for Food and Drug Control (Beijing, China). Azithromycin was dissolved in anhydrous ethanol; NAC was dissolved in distilled water. Anti-caspase-3, anti-Akt (pan), anti-p44/42 MAPK (Erk 1/2), anti-p38 MAPK, anti-PARP and p62 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3B antibody was purchased from Sigma (St. Louis, MO, USA). Azide-free anti-human CD261 (DR4) antibody was obtained from Diaclone (Besancon, France). Anti-DR5 antibody was obtained from ProSci (San Diego, California, USA). β-Actin (6G3) and GAPDH (1C4) monoclonal antibodies were purchased from AmeriBiopharma (Wilmington, Delaware, USA). Secondary antibodies included peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) (ZSGB-BIO, Beijing, China). Caspase inhibitor zVAD-fmk and RIP1 inhibitor necrostatin-1 were purchased from Selleck.cn (Houston, TX, USA). Chloroquine (CQ), acridine orange hemi (zinc chloride) salt (AO) and sulforhodamine B (SRB) were from Sigma.

For immunofluorescence assay, human OA chondrocytes were firstly fixed in 4% paraformaldehyde, followed by permeabilization with PBS containing 0.25% Triton X-100 for 15 min and then blocked with 5% BSA containing 0.25% Triton X-100 for 60 min at room temperature. Then the cells were incubated overnight at 4 °C with primary antibodies against protein HMGB1 (1:100; Abcam). After rinse for three times with PBS, the cells were then incubated with goat anti-rabbit IgG (H&L) conjugated with Alexa Fluor 594 (1:300; Zsgb Bio, China). After rinse for three times, the nuclei were counterstained with DAPI for 3 min. Images were taken by fluorescence microscopy (Carl Zeiss, USA). Total cells were counted in five random fields per culture dish and percentage of HMGB1 positive cells were determined.