Gb11119

TMJ samples were collected, using 4% paraformaldehyde and 10% EDTA for fixation and decalcification, and following embedded in paraffin, continuous mid‐sagittal sections of 4 μm were cut for subsequent staining. After dewaxing and gradient hydration, haematoxylin and eosin (HE) staining was performed to quantify the synovial inflammation.
Immunofluorescence staining was incubated with the designated primary antibody overnight at 4°C and then conjugated the primary antibody with a fluorescent secondary antibody to visualize staining, and 4′,6‐diamidino‐2‐phenylindole (DAPI) reagent was used to develop nuclei. Primary antibodies were as follows: rabbit anti‐ALPK1 (1:300, 19107‐1‐AP; Proteintech), mouse anti‐CD68 (1:500, 66,231‐2‐Ig, Proteintech), rabbit anti‐Vimentin (1:200, 10366‐1‐AP, Proteintech), rabbit anti F4/80 (1:200, GB11027, Servicebio) and rabbit anti‐INOS (1:500, GB11119, Servicebio).
Immunohistochemical staining was to incubate with the designated primary antibody at 4°C overnight, then with the designated secondary antibody for 30 min, and finally developed the colour with DAB (DAB‐0031, Bio technologies), followed by counterstaining of nuclei with haematoxylin. Primary antibodies were as follows: rabbit anti‐TNF‐α (1:400, 11948S, Cell Signaling Technology), rabbit anti‐IL‐1β (1:400, YT2322, Immunoway) and rabbit anti‐IL‐6 (1:400, A14687, ABclonal).

At 2 weeks post-injury, mice spinal cord tissues were fixed overnight in 4% paraformaldehyde. The samples were then permeabilized with 0.2% Triton X-100 (0.1%, Sigma-Aldrich) and blocked for 1h at 37°C in 1% BSA (6%, Bio-froxx, Germany) in 0.2% Triton X-100. After blocking, incubations were performed using specific primary antibodies (rabbit anti-CD206 [1:200, TD4149, Abmart], rabbit anti-iNOS [1:500, GB11119, Servicebio]) for 12 h followed by incubations with secondary antibodies for another 1 h. Images were captured using a fluorescence microscope (Olympus fluorescence microscope, Japan)
For quantitative real-time polymerase chain reaction, total mRNA was extracted from tissues (Life Technologies, USA) at 7dpi and 2 weeks post-injury using TRIzol-chloroform. cDNA was reversely transcribed with a PrimeScriptTM RT Master Mix (Takara, Dalian, China). PCR analysis was performed using a MiniOpticon Real-Time PCR system and Bio-Rad’s CFX manager software version 1.5. Finally, fold change was calculated as 2 to the -ΔΔCt power (2 -ΔΔCt). The qRT-PCR primers are summarized in Table 1.