Immunohistochemical Staining of CRCLM Sections

We performed immunohistochemical staining for formalin-fixed paraffin-embedded (FFPE) CRCLM sections as described in previous publications (17 (link), 20 (link)). Briefly, the sections were deparaffinized, hydrated, and exposed to antigen retrieval and endogenous peroxidase inhibition. The sections were then blocked with 5% goat serum buffer for 1 h at room temperature. The sections were incubated with primary antibodies overnight at 4°C. The sections were washed and exposed to secondary antibodies (Dako, Burlington, ON, Canada, anti-mouse: #K4001; anti-rabbit: #K4003) and incubated for 1 h at room temperature followed by staining with diaminobenzidine (DAB) substrate (Dako, Burlington, ON, Canada, #K3468). The stained sections were scanned and analyzed using (Aperio ScanScope XT System).
We used the following primary antibodies: RUNX1 1:200 (LS Bio, Seattle WA, USA, #LS-C353932), LY6G 1:1000 (Abcam, Waltham, MA, USA, # ab238132) and Ang1 1:1500 (Abcam, Waltham, MA, USA, #ab102015).