Figure 5
Analysis of vessel density in control and treated tumours. Tumours from control, o-OPN (dark-at-night conditions) or CLG (light-at-night conditions) treated mice were collected and cryosections or paraffin sections were prepared. Blood vessels were visualised with anti-CD-31 antibody or anti vWF antibody. (A and B) Cryosections from control (A) or o-OPN-treated (B) tumours were reacted with anti-CD-31 and visualised by DAB staining; sections were counterstained with haematoxylin. (C and D) Paraffin sections from control (C) and o-OPN (D) treated tumours were reacted with anti-vWF and visualised by DAB staining. Sections were counterstained with methyl green. Arrows indicate representative large and small vessels; bar=50 μm. (E) The number of CD-31-stained blood vessels in control, o-OPN- or CLG-treated tumours was manually counted in an area of 25 μm2. Tumours were collected on day 15 (NaCl, n=10 and CLG d15, n=5) or on day 19 (CLG day 19, n=8) after inoculation. o-OPN groups as in Figure 2. (F) The total CD-31-stained area per section was measured using a thresholding technique in Image J. Groups as in panel (E). (G) Total vessel area in the maximally stained field of each tumour section was measured in vWF-stained sections by using Image J to outline blood vessels. Groups as in Figure 2.
