Anti ox40

Tissue from freshly excised cSCC was obtained from patients during surgery at the Dermatology Department, University Hospital Southampton NHS Foundation Trust. The tissue samples (n=15 tumors) were snap frozen in liquid nitrogen, embedded in OCT medium, cryosectioned and immunostained as described previously (22 (link)). Primary antibodies included anti-CD3 (1:200, DAKO), anti-CD4 (1:50, Abcam), anti-CD8 (1:20, Invitrogen), anti-FOXP3 (1:20, eBioscience), anti-cytokeratin 16 (1:20, Thermo Scientific), anti-cytokeratin 17 (1:20, DAKO), anti CD31 (1:200, eBioscience), anti-CLA (1:200, Biolegend), anti-E-selectin (1:20, R & D Systems) and anti-OX40 (1:200, BD Biosciences). Fluorophore conjugated secondary antibodies comprised Alexa Fluor 488 goat anti-mouse IgG1a, Alexa Fluor 488 goat anti-rat IgM, Alexa Fluor 555 goat anti-rabbit IgG, Alexa Fluor 555 goat anti-rat IgG and Alexa Fluor 633 goat anti-mouse IgG2a (all from Invitrogen). Tissue sections were counterstained with DAPI (Sigma) before being mounted in Mowiol 4–88 (Harco) and imaged using a Zeiss Axioskop 2 fluorescence microscope or a Leica SP5 confocal microscope.

Ethical approval for the study was provided by the South Central Hampshire B NRES Committee (reference number 07/H0504/187). Archived formalin-fixed paraffin-embedded (FFPE) cSCC samples were obtained from University Hospital Southampton NHS Foundation Trust. Immunohistochemistry was performed as described previously (21 (link)), with microwave antigen retrieval performed in either 10 μM citrate or 1.6 μM EDTA buffers. Primary antibodies included anti-CD3 (1:200, DAKO), anti-CD4 (1:100, DAKO), anti-CD8 (1:20, Abcam), anti-CD25 (1:50, Novocastra), anti-FOXP3 (1:50, Abcam) and anti-OX40 (1:50, BD Biosciences), and biotinylated goat anti-mouse (1:200, DAKO) or swine anti-rabbit (1:400, DAKO) were used as secondary antibodies. For cell quantification, 5 representative images at 40× magnification were taken from each immunostained cSCC section and analysed using ImageJ software.