Dm6000 cfs microscope

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).

Images were acquired by Leica LAS AF 1.8.2 software with either an inverted Leica SP5 confocal system using a Leica DMI6000CS microscope or an upright Leica SP5 2 confocal system using a Leica DM 6,000 CFS microscope. Using the inverted microscope, images were acquired using a × 10 Leica Plan Apochromat objective with 0.40 numerical aperture for quantification and a × 20 Leica Plan Apochromat objective with 0.70 numerical aperture. Using the upright microscope, images were acquired using an HCX APO L20x objective with a 1.0 numerical aperture for still images and subsequent movies. Imaging of calvarium ranged from 60 to 100 μm. CFP (excitation 458 nm and emission 463–500 nm), GFP (excitation 488 nm, emission 493–556 nm) and DsRed2 (excitation 561 nm, emission 566–650 nm) were excited with an Argon/2 (458, 477, 488, 496 and 514 nm) and Diode pumped solid-state (561 nm) laser, respectively. The power used for dsRed visualization was 8–12% of the appropriate laser. Images were continuously captured in 1,024 × 1,024 or 1,024 × 512 format, with line averaging of 4 (∼10 or 5 s per scan, respectively) for up to 8 h. Multicolour imaging for CFP and GFP were captured sequentially.

Samples were washed in 1X PBS. The 1% (W/V) agarose was dissolved in PBS by heating the solution. Encapsulated organoids were placed in a 96-well plate, and PBS was removed. Agarose was added and allowed to solidify at 4 °C and subsequently embedded in paraffin. Paraffin blocks were sectioned at 4 µm thickness and stained with hematoxylin and eosin or used for immunohistological staining. For the latter, heat-induced epitope retrieval was performed using TRIS-EDTA buffer (pH = 8.0). Subsequently, slides were incubated in goat serum for 1 h before incubation of primary antibodies (listed in Table S3) overnight at 4 °C. Slides were washed using PBS before incubation (1 h, RT) with secondary antibodies (listed in Table S4). All slides were counterstained with DAPI (Vectashield anti-fade mounting medium with DAPI, Vectorlabs, Newark, CA, United States) and imaged on a Leica DM6000 CFS microscope with a LEICA TCS SP5 II confocal system. Data were processed and analyzed using ImageJ.