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.