Quantification of PCNA Immunolabeling

Paraffin sections of 3-μm thickness were stained with antibodies to α smooth muscle actin (α-SMA) (dilution 1:900, clone 1A4, Sigma, St. Louis, MO, USA), von Willebrand factor (vWF) (dilution 1:900, Dako, Hamburg, Germany) and proliferating cell nuclear antigen (PCNA) (dilution 1:200, sc-56, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Automated quantification of PCNA immunopositive labeling was performed using QuPath [29 (link)]. The software was trained to recognize PCNA-stained nuclei and positive-labeled cells using positive cell and subcellular detection modules. The number of cells with PCNA-positively labeling per μm² and the percentage of cells detected with PCNA immunolabeling were recorded and compared between all groups.