Dapi and anti hif1α fluorescein

In total, 5 ×10⁶ A549, MDA-MB-468, or MDA-MB-468/Luc cells (1:1 PBS:Matrigel) were injected subcutaneously into the hindflanks of nude mice (NCR nu/nu, Taconic). Tumors were allowed to form for 2–3 weeks. Nanoparticles (8.3 × 10¹² NP·kg^–1, 5% glucose) were injected via the tail vein into tumor-bearing nude or immunocompetent mice (BALB/c, Taconic). Tumors were harvested after 48 or 72 h and processed by the Swanson Biotechnology Histology Core Facility. Briefly, tumors were formalin-fixed, paraffin-embedded, sectioned (5 μm), deparaffinized, antigen retrieved, and stained using DAPI, biotinylated hyaluronic acid binding protein (Calbiochem), anti-CD44-FITC (Life Technologies), and streptavidin-Alexa Fluor 546 (Life Technologies) or DAPI and anti-HIF1α-fluorescein (R&D Systems). Slides were mounted and imaged using a Nikon 1AR Ultra-Fast Spectral Scanning Confocal Microscope. Whole-animal imaging was performed using a Xenogen IVIS Imaging System (Caliper) with d-luciferin (150 mg kg^–1 ip, PerkinElmer) as a bioluminescent substrate. These experiments were approved by the Massachusetts Institute of Technology Committee on Animal Care (CAC).