Immunohistochemical Analysis of Tumor Proliferation

Tumours harvested for paraffin embedding were fixed for 2 h in 4% paraformaldehyde (PFA) or 10% formalin and then embedded in paraffin using standard protocols. 4 µm sections underwent antigen retrieval and were stained using the DAKO Autostainer (K8012) as described previously^{14 (link)}. Sections were incubated for 30 min with Ki67 antibody (1:200) (DAKO M7240) in 5% BSA in Tris Buffered Saline followed by goat anti-mouse HRP (Abcam)(1:250) and staining with 3,3′-diaminobenzidine DAB (1 drop per ml). Haematoxylin staining was performed on all the slides to assist in distinguishing between tumour and chick nuclei. A total of 9 fields from 3 slides were counted per tumour and at least three tumours per condition were analysed.