D7137

Prior to termination of experiments, 0.5 mg of 2000-kDa-lysinated-rhodamine dextran (LRD) (D7139; Thermo Fisher SCIENTIFIC) or 2000-kDa-lysinated fluorescein dextran (LFD) (D7137; Thermo Fisher SCIENTIFIC), or 0.6 mg of 70-kDa-LRD (D1818; Thermo Fisher SCIENTIFIC) or 70-kDa-LFD (D1822; Thermo Fisher SCIENTIFIC) in 100 μl dH₂O was intravenously injected into the tail vein of each mouse. At 5-min or 15-min post injection, mice were euthanized by cervical dislocation or inhalation of high dose of isoflurane. Tumors and healthy tissues of non-tumor-bearing mice were removed and were fixed overnight with 4% PFA solution, followed by whole-mount immunostaining as described above. A rat anti-mouse CD31 (1:200) or a goat anti-mouse CD31 (1:200; AF3628; R&D SYSTEMS), a Cy5-labeled goat anti-rat secondary antibody (1:200; AP183S; Invitrogen), and an Alexa Fluor 555-labeled donkey anti-goat secondary antibody (1:200; A-21432; Invitrogen) were used for visualization of the vasculatures. LRD or LFD, and vascular positive signals were detected by confocal Microscopy (Nikon C1 Confocal microscope; Nikon Corporation, Japan) and three-dimensional images were collected. Vascular perfusion was quantified as a vessel area from the 2000-kDa-LRD/LFD positive signals per field and extravasation was quantified from the 70-kDa-LRD/LFD area extravasated from vessels per field.

Mice were anesthetized by an IP injection of a mixture of ketamine (100 mg kg⁻¹) and xylazine (20 mg kg⁻¹). The small intestine was surgically externalized, and the epithelium was exposed via a small incision in an area devoid of intestinal content. During the procedure, the epithelial tissue was constantly moistened by applying saline. The anesthetized mouse was placed on the microscopic stage and covered with a heated pad (37 °C) to maintain body temperature. Fixable fluorescent dextran conjugates of 3 or 2000 kDa in size (ThermoFisher D-3305 or D7137) were injected directly into the intestinal lumen via the incision, and the externalized epithelium was then positioned on a coverslip mounted on the stage above the objective and immobilized using custom-made holders. The blood flow was assessed visually by using the eyepiece and only regions close to blood vessels were imaged. The microscope used was a NIKON TiE inverted fluorescence microscope equipped with a Yokogawa CSU-21 spinning disc head and an Andor DU-897 camera. NIKON Elements software was used for image analysis.