Digital sight ds ue camera controller

PFA-fixed, paraffin-embedded tissue sections were rehydrated, and endogenous peroxidase was blocked by incubating the slides in 1.5% H2O2 solution (0.02 M citric acid, 0.06 M Na2HPO4) at RT for 15 min in the dark. Antigen retrieval was performed by boiling in Tris-EDTA (pH 9) or citric acid buffer for 20 min followed by slow cool down to RT. After blocking in 2% BSA in PBS at RT for 1 h, the sections were incubated with primary antibodies in blocking solution o/n at 4 °C (MPO, ab208670; CAV1, ab32577; both Abam). Following three washing steps in PBS, the sections were incubated with HRP-conjugated secondary anti-rabbit antibody (K4003, Dako EnVision+ System from Agilent) for 1 h at RT. After three washing steps in PBS, the signal was developed with DAB (Agilent, K3468). The sections were counterstained, dehydrated, and mounted using Eukitt medium (Sigma, 03989). For the detection of 8-OHdG (MOG-100P, JaICA), the Vector M.O.M immunodetection kit (BMK-2202) was used according to the manufacturer. Bright field images were acquired on a Nikon E600 microscope (Plan Fluor ELWD 20×/0.45 objective) with a Digital Sight DS-UE camera controller and DS-Ri1 camera (both Nikon) using Nikon software NIS Elements 4.0.

Fluorescence imaging was performed on a IMIC digital microscope (FEI, Type 4001) using the Polychrome V light source, an Orca-R² camera controller from Hamamatsu (C10600) and Live Acquisition software (FEI, version 2.6.0.14). Image analysis was performed using Offline Analysis software (FEI). Bright-field imaging was performed on a Nikon E600 microscope equipped with Nikon objectives (Plan Fluor ELWD 20x/0.45, Plan Apo 40x/1.0 Oil and 60x/1.40 Oil) using a Digital Sight DS-UE camera controller and DS-Ri1 camera (both Nikon). Image analysis was performed using Nikon software NIS Elements 4.0.