Anti-PCNA antibody [PC10] (ab29)

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Liver specimens collected at euthanization were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4 μm, deparaffinized, and rehydrated. Endogenous peroxidase was inactivated by incubation in 3% H2O2 in absolute methanol for 30 min. For antigen retrieval, the sections were microwaved in citrate buffer (pH 6.0) for 10 min. Sections were incubated overnight at 4°C with the primary monoclonal antibody against proliferating cell nuclear antigen (PCNA; ab29, Abcam, Cambridge, MA). After the cells were washed with PBS, immobilized antibodies were detected by the avidin-biotin-peroxidase technique (Vectastain ABC kit, Vector Laboratories, Burlingame, CA). 3,3′-Diaminobenzidine (Vector Laboratories) and hematoxylin (Sigma-Aldrich, St. Louis, MO) were used as the chromogen and the nuclear counterstain, respectively. The primary antibody was omitted as negative control. PCNA-positive and -negative nuclei were counted in five randomly selected fields for each sample, and each group included sections from at least three mice. PCNA expression was quantitated as the percentage of PCNA-positive cells.

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