Peroxidase conjugated streptavidin

The paraffin sections were stained as previously described [12] (link). Antibodies used were guinea pig anti-swine insulin (Dako Cytomation, Glostrup, Denmark), mouse anti-BrdU (Dako Cytomation), rabbit anti-survivin (Cell Signalling Technology), rabbit anti-C-peptide (Cell Signalling Technology, Danvers, MA, USA) and rabbit anti-ChromograninA (Dako Cytomation). Specificity of the survivin antiserum was confirmed by preabsorbtion with a survivin blocking peptide for 30 min (Cell Signalling Technology).
For light microscopy stainings the sections were incubated for 30 min at room temperature with a biotinylated secondary antibody (Zymed Laboratories, South San Francisco, CA, USA), rinsed and incubated with peroxidase-conjugated streptavidin (Zymed Laboratories). The sections were finally developed with 3-amino-9-ethyl-carbazole substrate (Thermo Scientific). For fluorescence microscopy, the secondary antibodies used were goat anti-guinea pig IgG (Alexa, A11076, Invitrogen, Paisley, UK), donkey anti-rabbit IgG (Alexa, A21206, Invitrogen) and donkey anti-mouse IgG (Alexa, A21202, Invitrogen). Nuclear staining was performed with DAPI (Vectashield with DAPI, Vector Laboratories, Burlingame, CA, USA).

Blood was centrifuged at 500 × g for 10 min. The resulting supernatants were stored at –20°C and used as plasma. An ELISA for the detection of IFN-γ or IL-10 in plasma was performed as described previously [37 (link), 38 (link)]. A rat anti-mouse IFN-γ mAb (clone R4–6A2; eBioscience) and a rat anti-mouse IL-10 mAb (clone JES5–16E3; eBioscience) were used as capture antibodies. A biotinylated, rat anti-mouse IFN-γ mAb (clone XMG1.2; eBioscience) and rat anti-mouse IL-10 mAb (clone JES5–2A5; eBioscience) were used as the detecting antibodies. The reaction was visualized by peroxidase-conjugated streptavidin (Zymed) and the substrate, 2,2’-azino-bis (3-etylbenzthiazoline-6-sulfonic acid) (ABTS) (Wako, Osaka, Japan). The absorbance of individual wells was determined using a Multiskan FC microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a wavelength of 414 nm. The concentrations of cytokines in plasma were calculated from standard curves prepared using known quantities of murine recombinant IFN-γ (Genzyme, Boston, MA, USA) and IL-10 (Pierce, Rockford, IL, USA). Purified antibodies for in vivo CD8⁺ cell-depletion (500 μg of anti-mouse CD8 mAb, clone 2.43, eBioscience) and neutrophil/macrophage-depletion (300 μg of anti-mouse Gr1 mAb, clone RB6-8C5; eBioscience, San Diego, CA, USA) were injected intraperitoneally into the mice.

An ELISA for the detection of IFN-γ or IL-10 in plasma was carried out as described previously [18 (link)]. A rat anti-mouse IFN-γ mAb (clone R4–6A2; eBioscience, San Diego, CA, USA) and a rat anti-mouse IL-10 (clone JES5–16E3; eBioscience) were used as the capture antibodies, and a biotinylated, rat anti-mouse IFN-γ mAb (clone XMG1.2; eBioscience) and rat anti-mouse IL-10 mAb (clone JES5–2A5; eBioscience) as the detecting antibodies. The concentrations of cytokines in plasma were calculated from standard curves prepared using known quantities of murine recombinant IFN-γ (Genzyme, Boston, MA, USA) and IL-10 (Pierce, Rockford, IL, USA). The reaction was visualized using peroxidase-conjugated streptavidin (Zymed) and the substrate 2,2’-azino-bis(3-etylbenzthiazoline-6-sulphonic acid) (Wako, Osaka, Japan).