Proliferating cell nuclear antigen (pcna)

Paraffin embedded sections were deparaffinised with Histoclear and rehydrated through graded ethanols. Heat induced antigen retrieval was performed in a microwave oven using Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, 0.05 % Tween 20, pH 9.0) for 20 min and the slides were allowed to cool down prior to blocking with 3 % H₂O₂. After blocking with 5 % (v/v) goat serum in phosphate buffered saline (PBS), sections were incubated overnight at 4 °C with the primary antibodies. After a PBS wash, the sections were incubated with secondary antibody at 37 °C for 30 min. Visualization of a positive reaction was by means of a peroxidase substrate solution containing 0.02 % (wt/Vol) H₂O₂ and 0.1 % (wt/Vol) 3,3′-diaminobenzidine tetrahydrochloride (ZSBIO, Beijing, China) in PBS to produce a brown color, then the sections were counterstained with hematoxylin. A negative control, with the primary antibody replaced by mouse IgG antibody, was always included. The primary antibodies used were anti-proliferating cell nuclear antigen antibody (PCNA) (Merck, Millipore, Darmstadt, Germany) diluted 1:400 to identify cell proliferation. PCNA positive cells were counted in 10 randomly selected × 200 high-power fields under a microscope (OLYMPUS FSX100). The PCNA index was calculated according to the following formula: number of PCNA positive cells/total cell count × 100 % [15 (link)].