Quantitative Immunohistochemistry for FRG1 Expression

From FFPE blocks, 5 µm thin sections were cut and deparaffinized by heating at 80 °C dry bath for an hour, then submerging the slides into xylene. Sections were rehydrated by a gradient of ethanol-100, 90, 70, 50%, and water. Heat-induced antigen retrieval was done in EnVision FLEX target retrieval solution (pH 9) high pH citrate buffer (DAKO, MN, USA) and blocked with EnVision FLEX Peroxidase blocking buffer (DAKO). Sections were incubated with primary and EnVision FLEX HRP secondary antibody (DAKO) for 2 hours and 30 minutes. We stained slides with EnVision FLEX DAB + Chromogen (DAKO) and counterstained them with Haematoxylin (Himedia, Mumbai, India). FRG1 expression was calculated using the Allred score (AS), as described previously [4 (link)]. Briefly, the Allred score was measured by combining the staining intensity of FRG1 protein in the cytoplasm and the percentage of cells stained positive for FRG1. Measurements of ‘staining intensity’ were categorized as weak “0–2”, moderate “3–6”, and strong “7–8”. FRG1 positive tumor tissue’s staining percentage were scored as “0” if 0%, “1” if 1%, “2” if 2–10%, “3” if 11–33%, “4” if 34–66% and “5” if ≥67%. Each sample was compared to the adjacent uninvolved tissue (if found) as a control.