Quantifying PADI2 Protein Expression in Tissue Samples

As described previously [21 ,22 (link),23 (link)], paraffin sections were deparaffinized and rehydrated [28 (link),29 (link),30 (link)]. Immunohistochemical (IHC) staining was conducted using a Super Sensitive™ Polymer-HRP IHC detection system (Biogenex, San Ramon, CA, USA). The slides were incubated with PADI2 polyclonal antibody (12110-1-AP, Proteintech, Rosemont, IL, USA) at a dilution of 1:50 at 4 °C overnight, followed by a wash, and then incubated with the peroxidase-conjugated secondary antibody (Abcam, Cambridge, UK), showing cytoplasm and nucleus staining. PADI2 was visualized using 3,3′-diaminobenzidine (DAB), and the slides were counterstained using hematoxylin. Oncological pathologists defined the protein expression scores using the multiplied values of stained intensity (0–2) and percentage of positive cells (0–100%). The PADI2 value was applied as a cutoff value to differentiate high or low expression.