Anti mouse cd31

As
previously described,^{18 (link),21 (link)} the tumor distribution of T-DM1-AF680
was analyzed using fluorescence microscopy at 1, 3, 5, and 7 days.
Briefly, nude mice were inoculated with 5 × 10⁶ NCI-N87
cells in the rear flanks and the clinical dose (3.6 mg/kg) of T-DM1-AF680
was administered via tail-vein injection once the longest axis of
the tumor was approximately 10–12 mm. Before euthanizing mice
at the aforementioned times, Hoechst 33342 (ThermoFisher Scientific,
H3570) was administered via the tail-vein at 15 mg/kg to label functional
vasculature in the tumor.^{31 (link)} After euthanizing
the mice, tumors were resected, flash frozen in OCT using isopentane
chilled on dry ice, and cut for histology on a cryostat (16-μm
slices). Before imaging, slices were stained with antimouse CD31 (BioLegend,
102402) conjugated with Alexa Fluor 555, and mouse antihuman IgG Fc
antibody (BioLegend, 409302) conjugated with Alexa Fluor 488. Microscopy
was performed using an upright Olympus FV1200 confocal microscope
equipped with a 20× objective and 405, 488, 543, and 635 lasers
(Figure S5). Tumor images were obtained
by stitching smaller images with the Olympus software. Images were
exported and analyzed using ImageJ image analysis software as described
previously.^{18 (link),21 (link)}