Three sections were chosen from the anterior, central, and posterior regions of the TMJ for immunohistochemical examination. Deparaffinized sections were immersed in 0.4s% hydrogen peroxide/methanol for endogenous peroxidase blocking. The sections were then incubated with 0.1s% trypsin for 20 min at 37°C and with 3 N HCl for 10 min as pretreatment. The pretreated sections were reacted with an anti-BrdU mouse monoclonal antibody (Bu20a; DakoCytomation, Copenhagen, Denmark) for 120 min, an anti-mouse-rabbit polyclonal antibody (DakoCytomation) for 60 min, and streptavidin-biotin horseradish peroxidase complex (DakoCytomation) for 30 min in turn. The immunoreaction was visualized using 3,3′-diaminobenzidine and the sections were lightly stained with hematoxylin. Normal mouse serum was substituted for the primary antibody in the negative control sections.
The BrdU-positive cells were counted in the IZ of the mandibular fossa, mandibular condyle, and articular disk between both ends of each structure in three immunostained sections from each animal at a magnification of ×200 under microscope. The labeling index was calculated for four control animals and four experimental animals at each of the three time points.
The BrdU-positive cells were counted in the IZ of the mandibular fossa, mandibular condyle, and articular disk between both ends of each structure in three immunostained sections from each animal at a magnification of ×200 under microscope. The labeling index was calculated for four control animals and four experimental animals at each of the three time points.