In the one animal per experimental group receiving fluorescent PFC nanoemulsion (CS-ATM DM Red, Celsense) spleens, tumors and livers were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA, Inc., Torrance, CA) and stored at −80 °C. Additional animals (N = 2 receiving CAR T cells and N = 2 receiving untransduced T cells) were sacrificed at day 2 for histopathology purposes. All tissues were cryosectioned (CM1950, Leica Microsystems Inc., Buffalo Grove, IL) at 10 μm thickness. Sections were fixed with 4% paraformaldehyde, stained for T cells using FITC anti-human CD3 (UCHT1, 1:500 dilution, Biolegend) and for nuclei using Hoechst dye (1:500) and then mounted. Tumor, spleen and liver sections were stained for macrophages with an Alexa 488 anti-mouse F4/80 antibody (BM8, 1:200 dilution, Biolegend)48 (link). Fluorescence images were acquired on an Axiovert 40 CFL microscope (Zeiss, Thornwood, NY) using a ×5 objective. Confocal images were acquired on a Leica SP5 2 confocal system with a Leica DM 6000 CFS microscope and a ×63 immersion objective. For direct cell counts in tumor, we used sections (two per tumor) stained against CD3 from five tumors total, three from day 2 and one each from days 7 and 14. We counted T cells in six high power fields per slice (×20 magnification, 120 high power fields total).
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