Alexa fluor 488 labeled goat anti human igg

To detect TF expression and vasculature, tumor tissues were fixed in 10% paraformaldehyde and sliced tumor sections (10 µm) were stained with 20 µg mL⁻¹ of ALT‐836 and 10 µg mL⁻¹ of CD31/PECAM‐1 antibody (MEC13.3; Novus), respectively. The washed sections were then stained with 5 µg mL⁻¹ of Alexa Fluor 488‐labeled goat anti‐human IgG (Invitrogen) and 5 µg mL⁻¹ of Cy3‐labeled donkey anti‐rat IgG (Jackson ImmunoResearch Laboratories, Inc.). After washing, the sections were mounted with the UltraCruz Hard‐set Mounting Medium containing 1.5 µg mL⁻¹ of DAPI (Santa Cruz Biotechnology). All the fluorescent images were acquired using a Nikon A1R confocal microscope.^[54] To detect the involvement of the adjacent structures, orthotopic tumors together with thyroid, trachea, muscle, and larynx were carefully resected at necropsy and used for H&E staining as previously described.^[51]