Quantifying Angiogenesis via Immunohistochemistry

Immunohistochemical staining for IGFBP-5 was carried out on 4-μm formalin-fixed, paraffin-embedded tissue sections. After antigen retrieval in 10 mM sodium citrate buffer (pH 6.5) in a microwave followed by blocking with an Avidin/biotin blocking kit (Vector Labs, Burlingame, CA, USA), tissue slides were stained with anti-IGFBP-5 antibody (Santa Cruz Biotechnologies, Dallas, TX, USA) and biotinylated anti-mouse secondary antibody (DAKO, Denmark). IGFBP-5 was detected with avidin-conjugated HRP (DAKO) and images were scanned with a Scan Scope (Aperio, Heidelberger, Germany). To quantify angiogenesis, microvessel density was determined by counting CD31-positive vessels as described previously46 (link). In brief, 8-μm thick sections of fresh-frozen tumor samples were fixed and incubated with anti-mouse CD31 antibody (Abcam, Cambridge, UK) at 4 °C overnight. CD31 staining was detected with an Envision detection system (DAKO).