Immunohistochemical Analysis of Cyclophilin A and CD147

Immunohistochemistry was performed in 4 μm paraffin sections. The tissue sections were dewaxed in xylene, rehydrated in graded ethanol, and rinsed in distilled water. Citrate buffer (pH = 6.0) was used to retrive the antigen, and 0.3% H₂O₂ was used to block endogenous peroxidase activity. After the blockade of nonspecific binding, tissue sections were incubated with the Cyclophilin A antibody (cat. no. 5360s; 1 : 150 dilution; Santa Cruz Biotechnology, Inc.) and CD147 antibody (cat. no. 13287; 1 : 400 dilution; Cell Signaling Technology, Inc.) for overnight at 4 centigrade. The secondary antibody was applied using the Envision Detection kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). The staining was performed by the application of diaminobenzidine tetrahydrochloride (DAB) for 2 min and hematoxylin for 1 min at room temperature. The stained sections were evaluated with a Nikon microscope in 10 independent fields at magnification, ×400. We followed the methods of Mu et al. [20 (link)].