Peroxidase igg fraction monoclonal mouse anti rabbit igg

Immunohistochemistry (IHC) was performed to validate candidate DEG potentially involved in the ancestry-associated discrepancy of TNBC, using FFPE samples. After deparaffinization, endogenous peroxidase blocking and antigen retrieval, samples were incubated 1h with primary antibody (Primary antibodies and respective dilutions are listed in the Supplementary Table S1), followed by 1h incubation with the secondary antibody (Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, light chain specific, code 211-032-171, Jackson ImmunoResearch). DAB (Bright-DAB Substract Kit, IL ImmunoLogic, VWR) was used for protein detection. Images were acquired using Axio Imager Z2 microscope (Zeiss). Protein expression was evaluated using Fiji ImageJ [42] (link) software and a semiquantitative approach to assign an H-score [43, (link)44] (link). The intensity of protein expression was measured using the scale 0-3, 0 being negative and 3 being very high expression. The H-Score was calculated by multiplying the intensity value with its area. Wilcoxon-Mann-Whitney test was used to compare mean Hscore of each protein between African-ancestry and White patients using the R function wilcox.test() and GraphPad Prism 6.01 software.