Goat anti mouse secondary antibodies

To assess the protein synthesis rate in single cells, puromycin incorporation was measured by immunofluorescence analysis. After paced for 6h, HL-1 cells were added with 10 μg/mL of puromycin (Solarbio, China) for 1 hour. Some cells were pretreated with 355 μM cycloheximide (BioVision, USA), a translation inhibitor, for 15 minutes serving as a negative control. Cells were fixed with 4% paraformaldehyde and allowed to stand at room temperature for 30 min. After washed with PBS, they were permeabilized with 0.1% Trion-X100 and blocked with 5% skim milk. The cells were incubated with primary antibody against puromycin (Merck millipore, USA) for 4 h, and then primary antibody against G3BP1 (Abcam, USA) overnight. Subsequently, the cells were incubated with goat anti-mouse secondary antibodies (Beijing Zhongshan Biotechnology Co., China) for 1 h, followed by staining with DAPI for 15 min. The images were captured by a laser confocal microscope (Zeiss LSM 510, Germany) with an excitation wavelength of 358 nm and emission 461 nm.

Immunohistochemical staining was performed with sections that had been deparaffinized and rehydrated. Slides were incubated with 0.3% hydrogen peroxide for 20 min before incubation with serum to reduce nonspecific binding. The slides were then incubated overnight with anti-CRP 1 : 200 (Abcam, USA), anti-IL-1β 1 : 100 (Bioss, China), anti-IL-10 1 : 150 (Bioss, China), anti-TNF-α 1 : 100 (Bioss, China), anti-CD68 1 : 100 (Abcam, USA), anti-inducible nitric oxide synthase (iNOS) 1 : 100 (Affinity, USA), and antireceptor activator of NF-κB (RANKL) 1 : 100 (Bioss, China). Then, the slides were washed and incubated with goat anti-mouse secondary antibodies for 30 min (Zhongshan Biotechnology, China), visualized with 3,3-diaminobenzidine tetrahydrochloride (DAB) substrate (Zhongshan Biotechnology, China), and counterstained with haematoxylin. Image-Pro Plus (6.0, Media Cybernetics, Rockville, MD) was used for the semiquantitative analysis, and protein expression was evaluated based on the integrated optical density (IOD).