The area of tumour stained by Hoechst 33342 as a percentage of the total area of tumour section was calculated using anaLYSIS (Soft Image Analysis).
Blood-vessel density was assessed by counting the total number of endomucin-positive vessels per mm across entire midline tumour sections from age-matched, size-matched tumours.
The sequentially recorded composite images of the tumour sections with detected perfused areas, hypoxia and total tumour vascular bed, respectively, were displayed using pseudocolours for each structure.
Initially, we found that the total number of blood vessels detected by endomucin staining per tumour section was similar for all treatment groups.
Light microscopy (400×) was used to count both PCNA positive proliferating cells and total tumour cells in three non necrotic areas of each tissue section as previously shown (Mukherjee et al, 1999a).
Proliferation and apoptosis were evaluated by counting Ki67-positive cells and apoptotic structures, respectively, from 3 to 5 random areas of each tumour section and calculated as a percentage of total cells.
For successful imaging, the contrast between tumour and surrounding tissue is of even higher importance than the total tumour uptake.
Areas of necrosis were measured using Image J and divided by the total tumour area/section to calculate percentage of necrotic area.
To visualise apoptosis, tumour sections were stained with cleaved caspase 3 antibody (1 : 400 dilution, CatNo9661 Cell Signaling Technology, Danvers, MA, USA) and positive staining areas were circled and compared to total tumour area using Image J software.
