most Figure 2 Immunoreactivity of the erythropoietin receptor. (A) Representative immunohistochemistry showing both cytoplasmatic and membrane staining of tumour cells and endothelial cells (arrow). Bar, 25��m. (B) Comparison of EPO-R immunoreactivity … Tissue pO2 and flow measurements Tissue oxygenation and laser Doppler flow (LDF) were measured with a fibreoptic probe combining fluorescence quenching with laser Doppler flowmetry (OxyLite? and OxyFlo?, Oxford Optronix, Oxford, UK) (Blackwell et al, 2003; Brurberg et al, 2004). A precalibrated fibreoptic probe was inserted 5mm deep into the tumour using a Seldinger technique; the probe was then withdrawn in 40 steps of 100��m each over a total distance of 4mm using a micromanipulator (model MN151, Narishige International Ltd, London, UK).

After each micromanipulator movement, measurements were started as soon as a stable reading was obtained. Tissue pO2 was sampled every 2s. Over this 4mm trajectory, oxygenation and LDF values were recorded separately for the tumour core (central 1�C2mm) and peripheral angiogenic rim (outer 1mm). Tissue pO2 was expressed in mmHg, whereas LDF was expressed in arbitrary units (a.u.). Immunohistochemistry Paraffin-embedded tissue samples were used for immunohistochemistry with the following antibodies: anti-EPO receptor (M-20) (sc-697, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Bcl-2 (sc-7832, Santa Cruz), anti-Bax (sc-7480, Santa Cruz), anti-VEGF (sc-7269, Santa Cruz), and anti-hypoxia-inducible factor-1 �� (HIF-1��) (sc-10790, Santa Cruz).

Paraffin-embedded sections were rehydrated by serial immersion in xylene and ethanol. After rinsing, the endogenous peroxidase was blocked with 0.3% hydrogen peroxide. The sections were subsequently incubated with a biotinylated secondary antibody, followed by incubation with a streptavidin�Cperoxidase complex (LSAB+kit, Dako, Heverlee, Belgium). The colour reaction was developed using 3-amino-9-ethylcarbazole substrate (Dako) as chromogen. Finally, the sections were counterstained with haematoxylin. Semiquantitative scoring was based on a method modified after Coppola et al (1998), with a scale ranging from 0 to 9. The scale was based on scoring of the fraction of positive cells (0: all cells negative; 1: <33% positive; 2: 33�C66% positive; 3: >66% positive) and the staining intensity (1: weak; 2: moderate; 3: intense).

Both scores were multiplied to a maximum score of 9. Scoring was performed separately on the tumour core and peripheral tumour rim. Microvascular density and diameter Microvascular density (MVD) was determined with a method modified after Weidner et al (1991). After incubating 5��m frozen slices with anti-CD31 antibodies (TLD-3A12, Serotec, Oxford, UK), the entire tumour section was scanned at low-power (objective, �� 4) to identify ��hot Dacomitinib spots’, which are the areas of highest neovascularisation.