Target retrieval system

PCNA expression in deparaffinized and rehydrated tissue sections was analyzed by immunostaining. Antigen exposure on sections was obtained by treatment for 20 min using target retrieval system (DAKO, CA, EUA). The activity of endogenous peroxidase was blocked by immersing sections in 3% hydrogen peroxide (H₂O₂) in 50% methanol. For reduction of nonspecific signals in the reaction, sections were then treated with blocking buffer (6% skim milk in PBS, pH 7.4) for 30 min at 37 °C. Incubation of sections with the primary antibody IgG2 anti-PCNA (Dako, CA, EUA), at a dilution of 1:1300, was carried out overnight at 4 °C. For conjugation with the secondary antibody, sections were treated with EnVision-HRP (horseradish peroxidase complex) (DAKO, CA, EUA) following the manufacturer’s specifications.
Sections were incubated with 3-amino-9-etil-carbazol (AEC) (DAKO, CA, EUA) for antibody conjugation with peroxidase, and then counterstained with Carazzi’s hematoxylin, also for antibodies with peroxidase. All stained tumor sections were imaged with light microscopy under 400× magnification. The analysis consisted of quantification of positive cells per field. The degree of PCNA expression was calculated as the percentage of antibody-labeled cells.