Envision system horseradish peroxidase kit

OLT-infiltrating neutrophils were detected using monoclonal rat anti-Ly6G Ab (BD Biosciences, San Jose, CA). Immunostaining signals were visualized with a labeled polymer in the EnVision + system horseradish peroxidase kit (Dako). Positively-stained cells were counted blindly (10 HPF/section). LC3B expression in OLT was detected using rabbit anti-LC3B Ab and signals were visualized with secondary Ab: Alexa Fluor 488 anti-rabbit IgG.

The expression of RXFP1 and GR (liver, heart, kidney, lung, esophagus) was examined using rabbit anti-RXFP1 Ab (Santa Cruz) and rabbit anti-GR mAb (Cell Signaling Technology, Danvers, MA). Immunostaining signals were visualized with a labeled polymer in the EnVision+ system horseradish-peroxidase kit (Dako, Carpinteria, CA). Liver infiltrating macrophages and neutrophils were detected with rat anti-CD11b Ab (BD Biosciences, San Jose, CA) and rat anti-Ly6G Ab (Bio-Rad, Hercules, CA), respectively. Signals were visualized with secondary Alexa Fluor 488 anti-rat IgG. Results were scored semi-quantitatively by blindly counting the number of positive cells in 10 HPF/section (x400).

The expression of RXFP1 (liver and heart) was examined using rabbit anti-RXFP1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA). Immunostaining signals were visualized with a labeled polymer in the EnVision+ system horseradish-peroxidase kit (Dako, Carpinteria, CA). Liver infiltrating neutrophils and macrophages were detected with rat anti-Ly6G Ab (BD Biosciences, San Jose, CA) and rabbit anti-CD11b Ab (Abcam, Cambridge, MA), respectively. Signals were visualized with secondary Alexa Fluor 488 IgG. Results were scored semi-quantitatively by blindly counting the number of positive cells in 10 HPF/section (×400).