Immunohistochemistry

enograft tumor from in vivo experiment was havested and fixed in 4% paraformaldehyde. Paraffin-embedded sections were used for haematoxylin-eosin (HE) staining and immunohistochemistry assays. For immunohistochemical staining, the sections were incubated with first antibody of SP1 (1:200, 9389, Cell Signaling Technology) or CCR7 (1:200, ab32527, Abcam) at 4℃ overnight. Then incubated in biotinylated Goat anti rabbit IgG at 37℃ for 30min, following by herseradish peroxidase (HRP) labeled avidin incubation. After visualization with diaminobenzidine (DAB), the sections were counterstained with hematoxylin and observed under 400x. Negative controls were obtained by omitting the primary antibodies. The expression of SP1 and CCR7 protein was semi-quantitatively measured by using Image Pro Plus 6.0 (Media Cybernetics, Inc., MD, USA)