Species appropriate irrelevant igg

Four micron-thick, formalin-fixed, paraffin-embedded tissue sections on de-identified slides were used for IHC analysis of Nodal expression. IHC staining was performed using a Dako Plus autostainer (Dako, Inc, Carpinteria, CA, USA) as previously described. 19 In brief, following antigen retrieval and blocking steps, sections were incubated in a goat antihuman Nodal antibody (LS-B3955; LifeSpan Biosciences, Seattle, WA, USA, 1:150) for mouse xenografts and the mouse monoclonal anti-Nodal antibody (ab55676, Abcam, 1:200) for human tissue sections for 60 min, followed by appropriate biotinylated secondary antibody (Biocare Medical, Concord, CA, USA), and then streptavidin-horseradish peroxidase (Thermo Scientific Lab Vision, Fremont, CA, USA). Immunostaining was detected using either 3,3′-diaminobenzidine (Thermo Scientific Lab Vision) or 3-amino-9-ethylcarbazole (Biocare Medical) peroxidase chromogen substrates. Sections were counterstained with hematoxylin (Biocare Medical). As a negative control, adjacent serial sections were incubated with species appropriate irrelevant IgG (Jackson ImmunoResearch Labs) at the same concentration as primary antibodies. Sections were reviewed and scored as previously described. 27