Mec13

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]

For immunohistochemical analysis of the thymus and lymph nodes (LNs), 6-μm thick frozen sections were fixed with acetone at −20°C and incubated with Alexa647-conjugated ERTR7 (Santa Cruz), Alexa488-conjugated anti-Lyve1 (223322; R&D Systems), and rabbit polyclonal anti-S1PR1 antibodies (H60; Santa Cruz), followed by incubation with Alexa546-conjugated goat anti-rabbit IgG antibody (Life Technologies). The images were captured using a confocal microscope (LSM 780; Zeiss). To detect Qa-2⁺ cells and tdTomato⁺ cells, the thymuses were fixed with 4% paraformaldehyde and then subsequently embedded in 4% low-melting agarose (Lonza) in PBS and cut into 200-μm thick sections using a vibratome (VT1200S; Leica). The sections were incubated overnight at 4°C in PBS containing 0.1% BSA and 1% Triton X-100, and Hoechst 33342 (Life Technologies), Alexa647-conjugated anti-Qa-2 antibody (695H1-9-9; BioLegend), DyLight550-conjugated anti-CD31 antibody (MEC13.3; Novus Biologicals), or APC-conjugated anti-CD31 antibody (MEC13.3; BioLegend). The images were captured using a spinning disk confocal microscope (Intelligent Imaging Innovations) and processed using Imaris software (Bitplane). Quantitation of the cortical and medullary area of the thymus was performed using ImageJ software (National Institute of Health).