Alexa fluor fluorescent dyes

Following surgical resection, tissues were fixed in 4% paraformaldehyde (4°C overnight). Tissues were cryoprotected in 30% sucrose/PBS then mounted in optimum cutting temperature medium. Tissue sections of 10 µm were cut on a Cryostat (Leica CM1860), incubated with blocking buffer (Dako, UK) for 1 hour, before primary antibodies were applied overnight (4°C). Antibody details are provided in online supplemental methods. Tissues were washed (PBS; 3×5 min), and species-specific secondary antibodies were conjugated to Alexa Fluor fluorescent dyes (1:400, Thermo Fisher Scientific, UK) applied for 1 hour, before washing (PBS; 3×5 min), mounting (Vectashield hard set mounting media, Vector Laboratories, USA) and cover-slipping. Controls with no primary antibody were used in all experiments to check for non-specific secondary antibody binding. Leica DM4000 epi-fluorescence microscope was used to visualise immunoreactivity of sections. To ensure uniformity of acquired images, all sections were orientated and cut in the same manner. Images were captured using MetaMorph software (Molecular Devices, UK) and prepared for figures using Photoshop (Adobe) and PowerPoint (Microsoft).

Cultures were fixed for 12 min at room temperature with 3.7% formaldehyde diluted in Dulbecco′s phosphate buffered saline (PBS) and then washed twice with PBS before incubation with primary antibodies for 24–48 h at 4 °C. The primary antibodies used for the cell culture protocols are listed in Table 1. Antibodies were pre-diluted in 0.2% Triton X-100, except for CD45, which was diluted in PBS only. We used anti-mouse/rabbit IgG (H+L) or anti-chicken IgY (H+L) conjugated to Alexa Fluor fluorescent dyes as secondary antibodies (Thermo Fisher Scientific).