Alexa fluor 488 labelled anti mouse

Tumor cells were incubated with monocyte-depleted PBMC of healthy donors (E:T 4:1), treated for 3 h and subsequently fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at -20°C. Cells were then blocked using 5% BSA, 0.2% Triton X-100, 0.1% Tween for 60 min, washed three times with PBST (PBS + 0.1% Tween20) and stained using a rabbit anti-human-α-Tubulin antibody (clone 11H10, Cell Signaling, Denvers, MA) and a murine anti-human-Perforin antibody (clone δG9, BD Pharmingen, Heidelberg, Germany), followed by an Alexa-Fluor 488 labelled anti-mouse and Alexa-Fluor 594 labelled anti-rabbit antibody (Invitrogen, Waltham, MA). Slides were mounted in fluorescent mounting medium; DAPI was used for counter-staining. Pictures were acquired using a Zeiss 800 inverse laser scanning microscope (Zeiss, Oberkochen, Germany) and images were processed using ImageJ (15 (link)).

Pancreatic tissue excised immediately after exsanguination was fixed in 4% (wt/vol.) formaldehyde and embedded in paraffin according to standard procedures. For co-staining of insulin and glucagon, mouse monoclonal anti-insulin (clone K36AC10, Sigma-Aldrich, Munich, Germany) and polyclonal rabbit anti-glucagon (Dako, Hamburg, Germany) antibodies were used. Alexa Fluor 546-labelled anti-rabbit (1:200) and Alexa Fluor 488-labelled anti-mouse (1:200; Invitrogen, Karlsruhe, Germany) were used as secondary antibodies. Nuclei were stained with DAPI.