Af568

Mouse tissue samples were immediately frozen in Tissue-Tek ® O.C.T. compound (SAKURA, Tokyo, Japan) or xed in 10% formalin and prepared in para n. OCT compound-embedded frozen samples were cut into 15-μm thick sections while para n-embedded samples were cut into 3-μm thick sections. Immuno uorescence analysis was performed to evaluate the area densities of the tumor microvessels, vascular normalization, and tumor hypoxia. The frozen tissue sections were xed in 5% paraformaldehyde for 15 min and the para n-embedded tissue sections were depara nized in xylene and hydrated with a graded alcohol series and distilled water. These tissue slides were blocked using 5% goat or donkey serum for 1 h. The sections were then incubated with the following primary antibodies: anti-CD31 (1:200, Angio-Protemie, Boston, MA), anti-α-smooth muscle actin (SMA) (1:100, Abcam, Cambridge, UK), or anti-pimonidazole (1:50, Hypoxyprobe Inc., Burlington, MA) antibody overnight at 4 °C.
The tissues were subsequently washed with phosphate-buffered saline (PBS) and incubated for 1 h at 25°C with a second antibody (diluted 1:400) conjugated to AF488, AF568, or Cy5 (Abcam). The cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The slides were observed under BZ-X800 uorescence (KEYENCE, Osaka, Japan) or laser confocal LSM 780 (Zeiss, Oberkochen, Germany) microscopes.