1 plus kit

Immunofluorescence assay of HIF‐1α and hypoxia marker was performed. 4T1 tumor bearing mice with tumor volume of about 200 mm³ were selected and i.v. injected with PpIX, P‐TH, and OP‐TH (2.5 mg kg⁻¹ PpIX), respectively. The tumor was dissected surgically 12 h post‐injection. After being embedded in an optimal cutting temperature compound (OCT), tumors were cut into tissue sections (7 µm) on a freezing microtome and attached to the slide. All tissues were incubated with primary anti‐Hif‐1α antibody for 2 h, and washed thrice with PBS. All samples were washed thrice with PBS containing 0.1% tween‐20 after being treated with secondary Alexa Fluor488‐conjugated goat anti‐rabbit IgG H&L antibody for 1 h. Finally, all samples were dyed with DAPI and recorded by CLSM. In the immunofluorescence study of hypoxia marker, the pretreatment of mice was similar to the above method. 6 h after the i.p. injection of pimonidazole HCl (Hypoxyprobe‐1 plus kit, Hypoxyprobe Inc., USA 60 mg kg⁻¹), the tumors were dissected and made into frozen tissue sections (7 µm). First, tumor tissues were treated with anti‐pimonidazole antibody (dilution rate 1:200, Hypoxyprobe‐1 plus kit). Next, horseradish peroxidase linked to goat anti‐mouse secondary antibody (dilution rate 1:200) was added according to the manufacturer's instructions. At last, all samples were stained with DAPI and observed under CLSM.

To evaluate the hypoxia alleviating ability of LIH in vivo, both xenograft and orthotopic CT-26 tumor model were used. The tumor bearing mice were randomly divided into three groups and were intravenously injected with saline, LI (100 μL, 6.0 mg total lipid per mL), and LIH (100 μL, 6.0 mg total lipid per mL and 10 mg Hb per mL) for three times (one injection per day), respectively. The tumors were dissected at 24 h after the last injection and were cut into 5 μm slices. For the detection of hypoxia in tumors, the tumor slides were stained using a commercial Hypoxyprobe-1 plus kit (Hypoxyprobe, Burlington, MA) following the protocol provided. For the detection of HIF-1α and VEGF, the tumor slides were incubated with polyclonal rabbit anti-HIF-1α antibody (dilution1:100) and VEGF antibody (dilution 1:100), respectively. After washing with PBS, the HIF-1α, and VEGF antibody was detected using FITC conjugated goat anti-rabbit immunoglobulin G, followed by observation using a confocal microscope.
To further investigate how long the alleviated tumor hypoxia state could maintain, the tumors were dissected at day 2, 4, and 7 after the last injection of LIH. The expression of HIF-1α and VEGF in tumor was determined using the methods as the mentioned above and the degree of hypoxia in tumor also was analyzed using Hypoxyprobe-1 plus kit.