Anti hexon antibody

Paraffin-embedded sections of mice brains (4 µm thickness) were were stained by hematoxylin/eosin method, or immunostained with anti-hexon antibody (1:2000; Merck Millipore; AB1056), biotinylated hyaluronic acid binding protein (1:400; Merck Millipore; 385911), anti-F4/80 antibody (1:400; Cell Signaling; 70076) and Iba1 (1:4000; Wako; 019-19741). For the immunohistochemical staining, Vectastain ABC kits (Vector Laboratories Inc., Burlingame, CA) were used according to the manufacturer’s instructions. Hyaluronic acid, F4/80 and Iba1 stained area was quantified using the Fiji platform^{18 (link)} in three different regions of the samples.

Tumours were fixed in 10% normal-buffered formalin prior to paraffin embedding (Leica tissue processor). Antibody staining was carried out with the automated Leica Bond Max (Leica, Milton Keynes, UK). Four-micron tissue slices were first incubated with anti-Hexon antibody (1:300 dilution, 1 h, clone 20/11, Merck Millipore, Watford, UK) and developed with a brown chromogen. Subsequently, epitope retrieval was performed at 60 °C for 20 min using Epitope Retrieval solution 2, followed by incubation with an anti-pimonidazole antibody (1:500 dilution, 30 min, P2627, Hypoxyprobe, Burlington, MA, USA) and red chromogen detection. For dual staining of VEGF RNA and Hexon antigen, the VEGF probe (#423161, ACD, Newark, CA, USA) was first hybridized and developed with a red chromogen according to the manufacturer’s protocol. Mouse IgG1 isotype (ab37355) and rabbit IgG isotype (ab171870) were used for staining controls (Abcam, Cambridge, UK). Slides were then stained for Hexon antigen as described above. Tumours were counterstained with haematoxylin, mounted and imaged with the Aperio Imagescope (Leica, Milton Keynes, UK).