Eclipse te2000 fluorescent microscope

For functional tumor vasculature assessment, tumor-bearing mice received an i.v. injection of 5 mg/kg FITC-lectin (Vector Labs, Burlingame, CA) 10 min prior to euthanasia. The tumor samples were frozen in OCT® medium, cryosectioned into 100 μm slices, and subsequently stained with anti-CD31 antibody. The fluorescence images were captured using a Nikon Eclipse TE2000 fluorescent microscope with Q-Capture software, and the analysis of microvessel density was performed using Image Pro Plus 5.1 (Media Cybernetics, Silver Spring, MD).

Cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 15 min for intracellular staining. Cells were then blocked with 3% bovine serum albumin (BSA) (Sigma) in PBS for 1 h at room temperature and incubated with primary antibodies diluted in 1% (w/v) BSA in PBS overnight at 4°C. Cells were washed with PBS three times and stained with secondary antibodies for 1 h at room temperature in the dark. The following primary and secondary antibodies were used: mouse anti-human OCT4 (AbD Serotec, UK), rabbit anti-human NANOG (Millipore), Alexa Fluor 488 anti-human TRA-1-60 antibody (BioLegend), mouse anti-human TRA-1-81 (Millipore), mouse anti-human Stage-Specific Embryonic Antigen-4 (SSEA-4) (Millipore), mouse anti-human NESTIN (Millipore), rabbit anti-human TUJ1 (Covance), mouse anti-human α-fetoprotein (Calbiochem), mouse anti-human actin (muscle) clone HHF35 (AbD Serotec), mouse anti-human PAX6 (Millipore), Alexa Fluor 488 γoat anti-mouse IgG (H+L), Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 568 goat anti-mouse IgG (H+L) (Life Technologies). Nuclei were stained with prolong gold DAPI with antifade (Invitrogen). Cells were visualized with an Eclipse TE2000 fluorescent microscope (Nikon, Japan).