2.3. Pimonidazole Immunostaining and Histology

Clinical tumour specimens and P2 PDX tumours were fixed in 10% neutral buffered formalin for 24–48 h, prior to long-term storage in 70% ethanol. Formalin-fixed tumour fragments were embedded in paraffin wax, cut on a microtome into 10 µM sections and mounted onto glass slides. The slides were deparaffinised and rehydrated by heating at 58 °C for 60 min and rinsing in xylene, ethanol and distilled water. Sections were stained with haematoxylin and eosin (Sigma-Aldrich) and visualised using a Zeiss Axiostar microscope and AxioVision 4.9.1 software (Carl Zeiss Microscopy, Oberkochen, Germany).

Slides with P2 PDX tumour sections from mice treated with pimonidazole were submerged in 10 mM citrate buffer (pH = 6.0) and placed in an Antigen Retriever 2100 pressure cooker (Aptum Biologics, Southampton, UK) for 2.5 h, followed by washing in Tris-buffered saline (TBS) and TBS with 0.1% Tween-20 (TBST). Sections were blocked for 1 h at 4 °C in 10% normal goat serum in TBST and stained overnight at 4 °C with pimonidazole-FITC antibody (mouse monoclonal antibody 4.3 11.3, NPI Inc., Burlington, MA, USA) diluted 1:50 in 5% normal goat serum in TBST. Sections were rinsed in TBST and counterstained with the nuclear marker Hoechst 33258 (Thermo Fisher Scientific). After further washing in TBST and TBS, slides were mounted with coverslips and imaged using a Zeiss Axio Imager Z2 microscope with a VSlide scanner (MetaSystems, Altlussheim, Germany). Two- to six-hundred images were taken at 20x magnification at 365 nm (Hoechst 33258) and 470 nm (FITC) using Metafer software 3.12.9 (MetaSystems) and stitched together using VSlide software 1.1.121 (MetaSystems) to create an image of a complete tumour slice. The pimonidazole-positive fraction was quantitated using ImageJ (v1.52e) by determining the immunostained area as a fraction of the viable tumour area, where necrotic regions were excluded. Necrosis was identified as a lack of Hoechst 33258 staining and was confirmed by histopathological examination of slides following de-coverslipping and staining for haematoxylin and eosin.