Quantitative Immunostaining for Protein Expression

A validation cohort, consisting of IUP (n = 25) and PUC with inverted growth (n = 16), was utilized independently from those used for the proteomic analysis. Immunostaining assays for SERPINH1 (1:200, sc-5293, RRID : AB_627757, Santa Cruz Biotechnology, Dallas, TX) and PYGB (1:2,000, HPA031067, RRID : AB_2673722, Sigma-Aldrich, St. Louis, MO) were conducted in IUP and PUC with inverted growth, using an automated BenchMark ULTRA System (Roche Diagnostics, Rotkreuz, Switzerland). The immunostained glass slides were digitally scanned using an Aperio Digital Pathology Slide Scanner AT2 (Leica Biosystems, Buffalo Grove, IL). Expression of SERPINH1 and PYGB was quantified by “H-score” [1 ∗ (% cells 1+) + 2 ∗ (% cells 2+) + 3 ∗ (% cells 3+)], with an interpretation ranging from 0 to 300 (33 (link)), using the QuPath platform for bioimage analysis (RRID : SCR_018257) (34 (link)). The area under the receiver operating characteristic (AUROC) was calculated using MedCalc version 20.019 (MedCalc Software Ltd, RRID : SCR_015044, Ostend, Belgium) and the optimal level of H-score with corresponding sensitivity and specificity were estimated on the basis of the Youden index.