2.2 Histological and Immunohistochemical
All the joint tissues were demineralized in 10% EDTA for 6 weeks and embedded in paraffin after gradient dehydration. Series mid-sagittal sections of 5μm were cut parallel to the condyle. After dewaxing in xylene and hydration in gradient alcohol, H&E, Safranin O staining were carried out according to themanufacturer’s protocol. Cartilage thickness was determined from H&E staining on the basis of the predecessors’ methods. A modified Mankin scoring system was used for cartilage OA score.
After dewaxing and hydration, the tissue sections were microwave antigen-retrieved in citrate solution. The sections were endogenous peroxidase activity blocked, following by serum blocking unspecific ligations. Rabbit anti-TLR2, TLR4, TLR8 were incubated overnight at 4°C severally, then reacted with anti-rabbit UltraSensitive S-P kit according to manufacturer’s protocol. For calculating the rate of positive cells, Image-Pro Plus 6.0 software was used to quantify the number of positive cells and total cells on selected cartilage area. Anterior, middle and posterior, 3 fields of each section were counted and averaged by two observers independently.