Gsl 1

Capillaries were visualized using lectin as described previously (Ballak et al., 2016 (link)). Briefly, air-dried sections were fixed with ice-cold acetone for 15 min, and blocked with 0.1% bovine serum albumin (BSA) diluted in Hepes for 60 min. Subsequently, the sections were treated with a peroxide solution for 30 min and incubated with biotinylated Griffonia (Bandeira) simplicifolia lectin (GSL I; Vector Laboratories, Peterborough, UK; 50 µg ml⁻¹ diluted in 1% BSA/Hepes) for 60 min. A 5 min wash was conducted between each step. Sections were then treated with avidin-biotinylated horseradish peroxidase (Vectastain ABC kit, Vector Laboratories) for 60 min, washed with Hepes, and incubated with horseradish peroxidase substrate diaminobenzidine (Vectastain DAB kit, Vector Laboratories) for 5 min. After a wash in distilled water, the sections were mounted in glycerol gelatin (Sigma-Aldrich, Aldrich, UK).

EL4 cells were s.c. injected, at a dose of 1 × 10⁶ cells/animal, into the right flank of four-to six-week-old C57BL/6 mice. Tumors were allowed to reach approximately 85 mm³ in size before DOX and HA treatments were started. Animals were distributed in different groups, and then s.c. treated on day 7 after tumor inoculation with saline, 20 μg/ml or 100 μg/ml of LMW HA, 1 μM of DOX, 1 μM of DOX plus 20 μg/ml of LMW HA or 1 μM of DOX plus 100 μg/ml of LMW HA. After 2 days, on day 9, mice were sacrificed, and tumors were removed, fixed in 4% formalin and embedded in paraffin. Before staining, 3-μm sections were deparaffinized and dehydrated. Slides were rinsed with PBS and dyed with DAPI 0.3 µg/ml plus fluorescein-labeled Griffonia (Bandeiraea) Simplicifolia Lectin I 20 µg/ml (GSL I, Vector Laboratories # FL-1101), which binds specifically to ECs in mouse tissues [72 (link)]. Furthermore, sections for histological analysis were routinely stained with hematoxylin/eosin. The sections were rinsed with PBS and then mounted on microscope slides. Micrographs of the stained sections were taken with a Nikon Eclipse E800 fluorescence microscope. Images were analyzed with the ImageJ 1.50b software package.

Sections were washed in 0.3 M PBST (3 × 10 min) and incubated with 5% NGS in 0.3 M PBST for 1 h at room temperature. Sections were then immunolabelled with rhodamine-labelled Griffonia simplicifolia lectin-1(GSL-1; Vector Laboratories RL-1102; 1:100) and incubated overnight at 4 °C. Sections were rinsed with 0.3 M PBST (3 × 10  min) and mounted with vectashield. Images were collected using a BioRad MRC 1000 laser scanning confocal microscope, 40X oil. The capillary density (number of capillaries per mm2) was determined using ImageJ, with a minimum of five non overlapping areas used for each sample.