Quantitative Analysis of CD276 Expression in NSCLC TMAs

The patient tissue microarray slide, including 73 cores of NSCLC patient tissue microarrays (TMAs) with duplication (LC1461), was purchased from US Biomax (Derwood, MD, USA). The TMA slides were processed following the procedure provided by the biomanufacturer and stained with recombinant rabbit anti-mouse/human CD276 antibody (AB134161, Abcam, Cambridge, UK) and horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA) to detect CD276 expression. The positive staining was visualized with DAB (3,3′-Diaminobenzidine) and imaged with a Vectra 3 automated quantitative pathology imaging system (Akoya Biosciences, Marlborough, MA, USA). Following our previously established TMA image analysis [29 (link),30 (link)], we used ImageJ software (Version 1.53t, National Institutes of Health, Bethesda, MD, USA) to quantitatively score CD276 expression with the Plugins-Analyze-RGB Measure. The expression score was calculated as red intensity/blue intensity with criteria of >1.1 as high and medium expression and <1.1 as low or no expression.