Proliferating cell nuclear antigen (pcna)

Pathology cases were selected from University of Kansas archives and formalin-fixed, paraffin embedded tissue blocks containing the most representative areas were selected for IHC. The following IHC antibodies were used: RAD6, RAD18, RPA70 (Abcam), and PCNA (Epitomics, Burlingame, CA, USA). Procedures were performed at room temperature using the Biocare IntelliPath autostainer. Epitope retrieval was by Biocare Borg Decloaker (BRCA-1) and citrate with pH 6 (PCNA, RAD6, RAD18, RPA70). Titers used were 1:500 (BRCA-1), 1:600 (PCNA), 1:3000 (RAD6), 1:300 (RAD18), and 1:800 (RPA70), with an incubation of 30 min for all antibodies. Detection was with Dako Evision FLEX HRP (BRCA-1), Biocare Mach 2 Rabbit HRP-polymer (PCNA, RAD6, RPA70), and Dako Envision+ LP, Mouse (RAD18). Slides were counterstained with hematoxylin and permanently mounted. Whole slide images were then analyzed by digital image analysis using Aperio (Leica Biosystems), and results were documented as an expression score incorporating both total percent positivity and intensity of staining [(percent positivity x staining intensity)/100]. Statistical analysis (one-way ANOVA and Tukey-Kramer test) was performed using Microsoft Excel with Real Statistics Resource pack and an alpha level of 0.05.