Ld plan neofluar 20 0.4 objective

Directly and indirectly labeled monoclonal antibodies as listed in online supplementary table 2 and DAPI as a nuclear marker were used. An isotype was used as negative control. In brief, after incubation with the primary antibodies overnight at 4°C, an appropriate secondary fluorescence-labeled antibody was applied for 30 min at room temperature, followed by staining of DAPI nuclear marker. For immunohistochemical staining, antibodies were mixed with 2% bovine serum albumin in PBS and applied overnight to sections in a humid chamber at 4°C. For visualization of the cells, AEC was used as chromogen. Sections were counter-stained with hematoxylin. For evaluation of staining results, images of whole tissue sections (one section per patient) were acquired using a Z1 Axio Observer microscope equipped with a LD Plan-Neofluar 20×/0.4 objective (Zeiss). For leucocyte evaluation, the tumor normal interface (5 mm on tumor and normal zone) was defined as regions of interests (ROI). For γH2AX staining, only tumor tissue was selected as ROI. ROI were automatically quantified using TissueFAXS/TissueQUEST image analysis software (TissueGnostics GmbH).