Hif 1α

Mice were cardiac perfused with 4% PFA (Daejung Chemicals) in PBS and the brain was harvested and made into frozen sections, followed by fixation using ice-cold 100% methanol for 10 min at the room temperature. Sections were then incubated with antibodies against Iba-1 (goat anti-mouse Iba-1 polyclonal antibodies, Abcam), CD68 (rat anti-mouse CD68 monoclonal antibodies, Abcam), HIF-1α (goat anti-rabbit HIF-1α antibodies, Novus), NeuN (rabbit anti-mouse NeuN antibodies, Abcam), DCX (rabbit anti-mouse DCX antibodies), Ki67-FITC (Biolegends), caspase-3 (rabbit anti-mouse antibodies, Abcam), or cleaved caspase-3 (CC3, rabbit anti-mouse CC3 antibodies, Cell signaling technology) antibodies for overnight at 4°C. Secondary antibodies of species-matched IgG conjugated with Alexa 488 and/or Alexa 546 (Thermo Fisher Scientific) were incubated for 1 hr at the room temperature. Sections were finally mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and examined with Zeiss Axio Scope with EC PLAN NEOFLUAR at 10×, 20×, and 40× objective lenses. Digital images were taken using AxioCam HRM camera and processed with AxioVision 4.8 software using 20× objective fluorescence microscope as described above. Images were evaluated at least three independent areas per mouse. Area densities were calculated by Image J software (National Institutes of Health).

Four micrometer skin sections from formalin fixed-paraffin tumors were rehydrated in PBS buffer and permeabilized by incubation for 10 min with 0.5% Triton X-100. Then, slides were incubated for 15 min with 0.5% bovine serum albumin and 5% goat serum, and for 60 min at 37 °C with the rabbit polyclonal anti-human Survivin antibody (1:50, Novus, Bloomington, MN, USA) or HIF1α (1:500, Novus, Bloomington, MN, USA), VEGF (1:50, Thermo Fisher Scientific, Waltham, MA, USA), CD51 (1:50, Abcam, Cambridge, UK). After four washes in PBS, samples were incubated for 60 min with the anti-rabbit secondary antibody, Alexa Fluor 546 (1:100, Thermo Fisher Scientific, Waltham, MA, USA). Fluorescent specimens were analyzed by confocal scanning laser microscope (Leica TCS SP2) and positive cell counts were performed by ImageJ software analysis.

Mice were culled at indicated time points post-microbeads' administration or 14 days post-LLC injection as described (Branco-Price et al., 2012 (link)). Lungs were fixed in 4% paraformaldehyde and embedded in paraffin and cross-sections (7 μm) were cut at 200 μm intervals throughout the lung. Contiguous sections were stained with haematoxylin and eosin (H&E, Sigma Aldrich, UK), Martius Scarlet Blue (MSB, Sigma Aldrich, UK), and for HIF1α (Novus Biologicals, UK), HIF2α (Novus Biologicals, UK), and Mac2 (BioLegend Ltd, UK) as previously described (Evans et al., 2010 (link), 2014a (link),b (link),c (link); Saha et al., 2013 (link)).