Image Analysis of Aggrecan Degradation in Articular Cartilage With Formalin-Fixed Samples
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Many studies in arthritis research require an evaluation of the cellular responses within the joint and the ensuing matrix degradation in articular cartilage. The early histochemical/histological scale of Mankin (1 ) has been widely used but recently challenged as insufficient (2 ). Imaging techniques such as microscopic magnetic resonance imaging (MRI) (3 ), polarized light microscopy (3 ), atomic force microscopy (4 ), and infrared spectral analysis (5 ) have opened new approaches to evaluating cartilage structure. Histological methods now include in situ hybridization for cell-specific gene expression and immunohistochemistry for the spatial organization of cartilage proteins and their processed forms.
This chapter details of a method for immunohistochemical analysis of aggrecan degradation in articular cartilage samples which have been prepared by standard methods of formalin fixation and paraffin embedding. The procedure focuses on the application of antibodies (e.g., anti-ADAMTS4, anti-MT4MMP) which detect some of the proteinases most likely involved, and anti-NITEGE which detects the terminal product of the aggrecanase-mediated cleavage of aggrecan at Glu392-Ala393 (bovine, human, dog, rat, pig, sheep, horse, mouse) or Glu393-Ala394 (chick).
