4.8. Immunohistochemistry (IHC)

The expression of CD34 (ab8158, abcam) was detected by immunohistochemical staining. In brief, after rehydrated in water, the paraffin sections were placed in citric buffer (pH 6.0) and treated in a microwave. Afterwards, the sections underwent blocking with 5% normal horse serum+ 1% normal goat serum and then primary antibodies were applied (incubated at 4 °C overnight). Then secondary antibodies from HRP-Rat IgG (ab97057, abcam) were applied. Signal was developed with DAB (Sigma-Aldrich). All procedures were performed in accordance with the manufacturer’s recommendations. Apoptosis was determined on paraffin-embedded sections (4-mm thick) by the TUNEL assay method with the use of an ApopTag Peroxidase In Situ Apoptosis Detection kit (S7100, Millipore, CA, USA), according to the manufacturer’s instruction. For quantification, the number of TUNEL-positive nuclei per field was counted manually. Three representative fields per tissue sample from each mouse were scored, yielding a ratio apoptosis value. Graphs represent the mean values of ratio apoptosis (mean ± standard error) compared to control. The IHC images of the tumor sections were obtained using the Aperio imaging system (Leica Biosystems, Wetzlar, Germany).