Microscopy digital camera system

After routine deparaffinization and rehydration, the sections underwent antigen retrieval in 0.125% trypsin-EDTA (Solarbio, Beijing, China) for 20 min at 37°C. Histostain™-Plus kits (ZSGB-Bio, Beijing, China) were used according to the manufacturer's recommendations. After incubation in goat serum, sections were incubated with the primary antibodies against TLR4 and IL-1β (1 : 1000, Cell Signaling, Beverly, MA, USA), respectively, overnight at 4°C. After rinsing with 0.01 M PBS, the sections were exposed to goat anti-rabbit secondary antibody (ZSGB-Bio, Beijing, China) for 30 min at 37°C, then were exposed to a solution of horseradish peroxidase-conjugated avidin-biotin complex (ZSGB-Bio, Beijing, China) for 20 min at 37°C. Then, sections were visualized with 0.1% 3, 30-diaminobenzidine dihydrochloride (DAB) (ZSGB-Bio, Beijing, China), and the sections were counterstained with hematoxylin. The digital images were captured using a microscopy digital camera system (Olympus, Tokyo, Japan). The results were evaluated semiquantitatively using the Image-Pro Plus 6.0 software. Five sections per rat were assayed in high power, and the mean optical density (MOD) was measured, respectively. The mean of MOD of five sections was seen as relative protein expression of this rat.

According to the manufacturer's recommendations, in all the groups, the expressions of SDF‐1 and IL‐1β in the paraffin sections were examined using an indirect immunohistochemical approach. First, the tissue antigens were repaired using 0.125% trypsin in a 37°C incubator for 20 min, following which the experimental sections were incubated in goat serum and were then incubated overnight at 4°C with the primary antibodies against IL‐1β (1:1000, Cell Signaling) and SDF‐1 (bs‐4938R, rabbit polyclonal, Bioss, USA, 1:1300 dilution), respectively. Subsequently, we used a secondary antibody kit (Zhongshan‐Golden‐Bridge‐Biotechnology). Following this, they were exposed to a solution of horseradish peroxidase‐conjugated avidin‐biotin complex (ZSGB‐Bio, Beijing, China) for 20 min at 37°C. Next, the sections were visualized with 0.1% DAB (3, 30‐diaminobenzidine dihydrochloride) (ZSGB‐Bio) and were counterstained with hematoxylin. We used a microscopy‐digital camera system (Olympus) to acquire digital images. The immunostaining of SDF‐1 and IL‐1β in each type of the TMJ cells was semiquantitatively evaluated using the Image‐Pro Plus 6.0 software. The mean of the mean optical density (MOD) of three sections was considered as the relative protein expression in each rat.