Figure 3: Increased vessel diameter and pericyte coverage in Tlr^−/− mice.

(a–c) Frozen sections of s.c. tumours dissected at day 6, day 10 or 14 after injection were stained with αCD31 antibody, to assess the vascular system of tumours of 8- to 16-week-old C57BL/6 WT and Tlr3/7/9^−/− mice. (a) Representative examples of tumours at day 10 und day 14. Scale bar, 100 μm. (b) For analysis of vessel diameter, vessels were assessed manually in × 200 magnified images and diameter was quantified using ImageJ software (n=3). P-value (t-test) is indicated (*P⩽0.05). Single values and mean (horizontal line) are shown. (c) Quantification of vessel number was performed by manual observer assisted counting of four × 200 magnified randomly selected images per sample (n=6–7). Single values and mean (horizontal line) are shown. Results were considered significant at *P⩽0.05 (t-test). (d) Twelve- to 16-week-old C57BL/6 WT and Tlr3/7/9^−/− mice were perfused and s.c. tumours were surgically removed 10 days after injection of MOPC^−eGFP tumour cells (green). Before tumour removal, vessels were stained in vivo by injection of Alexa Fluor 647-coupled αCD31 antibody (red). Tumour tissue was cleared and analysed by light sheet microscopy. Three-dimensional images were created using ImageJ and Imaris software. Shown are two representative examples of tumours of WT and TLR-deficient mice. Scale bar, 600 μm. (e) Three-colour immunofluorescence of explanted tumours. A representative example of CD31 (vessels) and caspase 3 (cell death) of d10 and d14 tumour sections from 8- to 16-week-old C57BL/6 WT and Tlr3/7/9^−/− mice is shown. Dapi (4,6-diamidino-2-phenylindole; blue) was used to visualize nuclei. Scale bar, 50 μm. (f) Pericyte staining of vessels. A representative example of CD31 (vessels) and desmin (pericytes) of d14 tumour sections from 8- to 16-week-old C57BL/6 WT and Tlr3/7/9^−/− mice is shown. Dapi (blue) was used to visualize nuclei. Scale bars, 100 μm. (g) Desmin-positive vessels from n=3 mice at day 10 and day 14 were quantified by manual counting of up to five fields per tumour, including 100 vessels in total, using the mean of three tumour section per tumour (top, middle and base) of concordant distance. Single values and mean (horizontal line) are shown. Three stars (***) indicate statistical difference P⩽0.0005 (t-test).