Paraffin sections were pretreated with xylene and dehydrated in ethanol. Antigen retrieval was performed using citrate or EDTA buffer for 15 min at subboiling temperature in a microwave, followed by blocking of endogenous peroxidase activity. Primary antibody was incubated overnight at 4°C, and secondary biotinylated antibody was detected with streptavidin–horseradish peroxidase (HRP) (Vector Laboratories). Sections were developed using a diaminobezidine Peroxidase Substrate Kit (Vector Laboratories).

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