Envision flex hrp secondary antibody

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.

IHC reactions were performed on 4 µm thick TMA sections placed on Superfrost Plus slides (Menzel Gläser, Braunschweig, Germany). The reactions were performed using the EnVision Flex System (Dako, Glostrup, Denmark). Deparaffinization, rehydration, and antigen retrieval (97 °C, 20 min) were performed in low-pH EnVision FLEX Target Antigen Retrieval Solution (pH = 6) using the PT Link (Dako, Glostrup, Denmark). Dako Autostainer Link48 was used for performing IHC reactions (Dako, Glostrup, Denmark). Endogenous peroxidase activity was blocked by a 5 min incubation with EnVision FLEX Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark). The sections were incubated for 20 min with anti-ZYX monoclonal antibody (1:50, 2D1 clone, catalogue no. sc-293448, Santa Cruz Biotechnology, Dallas, TX, USA) followed by EnVision FLEX + MOUSE LINKER for 15 min. Next, the sections were incubated for 20 min with EnVision FLEX/HRP secondary antibody (Dako, Glostrup, Denmark). DAB+ Chromogen (Dako, Glostrup, Denmark) was used to visualize the reaction. Hematoxylin was used to visualize cell nuclei according to the manufacturer’s instructions (Dako, Glostrup, Denmark).