Fitc mab1

For immunohistochemistry analysis, 4T1 tumor-bearing mice were intravenously injected with TiH_1.924-PVP (20 mg·kg⁻¹). At 8 h p.i., tumors on these mice were exposed to the 1064-nm laser irradiation for 20 min with their temperature maintained at ~45 °C. Then immediately, tumors were surgically excised for hypoxia staining assay using the Hypoxyprobe-1 plus kit (Hypoxyprobe Inc) following the standard protocol^{61 (link),62} . Anti-pimonidazole mouse monoclonal antibody conjugated with FITC (FITC-Mab1, Hypoxyprobe Inc.; Cat. No.: HP2-100Kit; Lot No.: 04-11-19; Clone: 4.3.11.3; Dilution: 1:200) and Alex 488-conjugated goat anti-mouse secondary antibody (Jackon Inc., Cat. No.: 115-545-003, Lot No.: 146108, RRID: AB_2338840; dilution: 1:200) for hypoxia staining. Rat anti-CD31 mouse monoclonal antibody (Biolegend Inc., Cat. No.: 102402, Lot No.: B226360, Clone: 390; dilution: 1:100) and Rhodamine-conjugated donkey anti-rat secondary antibody (Jackon Inc. Cat. No.: 712-025-150, Lot No.: 147079, RRID: AB_2340635; Dilution: 1:200) for blood vessel staining.

Hepatocytes and liver were lyzed with RIPA lysis buffer (Millpore, MA, USA). The concentration of total protein in the supernatant was measured by BCA kit (Thermo Fisher Scientific, MA, USA). The primary antibody HIF-1α (Novus Biologicals, 1:1000), HIF-2α (Novus Biologicals, 1: 1000), α-Tubulin (Cell signaling technology, 1: 1000), β-actin (Cell signaling technology, 1:1000), FITC-MAb1(Hypoxyprobe, Inc,1:10,000) were incubated in 4 °C overnight. The secondary antibodies (goat anti Rabbit, 1:5000, goat anti mus, 1:10,000, rabbit anti-FITC HRP1:10,000) were incubated 1 h in room temperature. ECL luminescence liquid (Lulong, Xiamen, China) was used while the light emitted was captured by the machine followed by the pictures being saved.