As previously described7 (link),
32 (link), 36 (link), the tumor distribution of fluorescent
antibody was analyzed using fluorescence microscopy 24 hours post-injection.
Briefly, 0.7 nmol fluorescent antibody (or 0.067 nmol of J591 anti-PSMA IgG) was
administered via tail-vein injection once the tumor volume was
~250mm3. The animal was imaged 24 hours post-injection.
Hoechst 33342 (ThermoFisher Scientific) was administered 15 minutes before
euthanasia via the tail-vein at 15 mg/kg to label functional vasculature in the
tumor. Tumors were then resected, flash frozen in OCT using isopentane chilled
on dry ice, and sectioned for histology on a cryostat (10-μm slices).
Before imaging, tumor slices were stained for 30 min with anti-mouse CD31
(BioLegend, 102402) conjugated to Alexa Fluor 555. Microscopy was performed
using an upright Olympus FV1200 confocal microscope equipped with a 20×
objective and 405, 543, and 635 lasers. High resolution tumor images were
obtained by stitching smaller images with the Olympus software. Images were
exported and analyzed using ImageJ image analysis software as described
previously7 (link), 32 (link), 36 (link).
32 (link), 36 (link), the tumor distribution of fluorescent
antibody was analyzed using fluorescence microscopy 24 hours post-injection.
Briefly, 0.7 nmol fluorescent antibody (or 0.067 nmol of J591 anti-PSMA IgG) was
administered via tail-vein injection once the tumor volume was
~250mm3. The animal was imaged 24 hours post-injection.
Hoechst 33342 (ThermoFisher Scientific) was administered 15 minutes before
euthanasia via the tail-vein at 15 mg/kg to label functional vasculature in the
tumor. Tumors were then resected, flash frozen in OCT using isopentane chilled
on dry ice, and sectioned for histology on a cryostat (10-μm slices).
Before imaging, tumor slices were stained for 30 min with anti-mouse CD31
(BioLegend, 102402) conjugated to Alexa Fluor 555. Microscopy was performed
using an upright Olympus FV1200 confocal microscope equipped with a 20×
objective and 405, 543, and 635 lasers. High resolution tumor images were
obtained by stitching smaller images with the Olympus software. Images were
exported and analyzed using ImageJ image analysis software as described
previously7 (link), 32 (link), 36 (link).
