D p x neutral mounting medium

For histological analysis, cryosections (6–10 µm thick) were prepared using a Leica CM1850 cryotome (Wetzlar, Germany). Sections were collected onto warm, charged Menzel Superfrost slides (Thermo Fisher), fixed in ice-cold 100% acetone, air dried and stored at −80 °C. For immunohistochemistry, tissues were fixed in paraformaldehyde and dehydrated through a graded series of ethanol and xylene, before being embedded in paraffin. Sections (5 µm) were mounted on to glass Menzel Superfrost slides (ThermoFisher Scientific). Immunohistochemistry was performed using antibodies for the proliferation marker Ki67 (rabbit anti-human Ki67, Abcam, Cambridge, UK) and for the infiltration of murine blood vessels using rabbit anti-murine CD31 antibody (Abcam). Tissue sections were incubated with HRP-polymer conjugates (SuperPicture, Thermo Fisher Scientific), and incubated with the chromagen diaminobenzidine (DAB) (Dako, Glostrup, Denmark), as per manufacturer’s specifications. Slides were counterstained with Mayer’s hematoxylin, dehydrated, and mounted with coverslips using D.P.X neutral mounting medium (Sigma Aldrich). All sections were counterstained with Mayer’s hematoxylin (Sigma Aldrich) and mounted with coverslips using D.P.X with Colourfast (Fronine, ThermoFisher Scientific).

Tissue sections were stained with haematoxylin and eosin solutions before dehydration and mounting with D.P.X. neutral mounting medium (Sigma-Aldrich). Digital images were captured with an Eclipse E400 microscope and DXM 1200 digital camera (Nikon, Kingston upon Thames, Surrey, UK), analysed using ImageJ software (Wayne Rasband, US National Institutes of Health, Bethesda, MD) and calibrated using a 100 μm gradient slide. A minimum of three images were captured per section with a minimum of 8 sections prepared per individual, with each section being 5 sections apart within the block. All images were captured under the same conditions. The villus height and the crypt depth measurements were determined blindly taken from an average of sixteen well oriented crypt-villus units per patient.

Tissue was processed and embedded in paraffin before sectioning (5 μM sections). Immunohistochemistry was performed on tumour sections to investigate the expression of the proliferation marker Ki67 (rabbit anti-Ki67 primary antibody, undiluted, Roche, Basel, Switzerland) and CD31, a marker of angiogenesis (rabbit anti-mouse CD31 primary antibody diluted 1:50 in antibody diluent; Abcam, Cambridge, UK). Sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted with coverslips using D.P.X neutral mounting medium (Sigma-Aldrich) and observed using an Olympus BX41/702 microscope (U-CMAD3) (Shinjuku, Tokyo Metropolis, Japan).