Bz x710 fluorescent microscope

Images were collected using a Keyence BZ-X710 fluorescent microscope configured with 3 fluorescent channels (FITC, Cy3, Cy5) and equipped with Nikon PlanFluor 40x NA 1.3 oil immersion lens. Imaging and washes were iteratively performed automatically using a specially developed fluidics setup (Figures S7A–S7C). Images were subject to deconvolution using Microvolution software (http://www.microvolution.com/). The staining patterns of 28 DNA-conjugated antibodies were acquired over 14 cycles of CODEX imaging and overlaid with 2 additional fluorescent antibodies, CD45-FITC and NKp46-Pacific Blue and a DRAQ5 nuclear stain (Figure 3A and low-resolution views in Video S2). Each tissue was imaged with a 40x oil immersion objective in a 7x9 tiled acquisition at 1386x1008 pixels per tile and 188 nm/pixel resolution and 11 z-planes per tile (axial resolution 900 nm). Images were subjected to deconvolution to remove out-of-focus light. After drift-compensation and stitching, we obtained a total of 9 images (one per tissue) with x = 9702 y = 9072 z = 11 dimensions, each consisting of 31 channels (30 antibodies and 1 nuclear stain).