Colorview camera

Oxidative DNA damage was measured by the formamidopyrimidine DNA glycosylase (FPG)-modified alkaline Comet Assay. Briefly, monocytes were co-cultured with PMA-activated macrophages or co-treated with 100 ng/ml PMA for 1 h. Monocytes were harvested, embedded in 0.5% low melting point agarose and transferred onto agarose-precoated slides. The slides were incubated in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X, pH 10) for 1 h at 4°C. Slides were equilibrated 2 x 5 min in buffer F (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2% BSA, pH 8.0) at RT. FPG was diluted in buffer F and 50 μl was added to the slides and incubated in a humid chamber at 37°C for 45 min. DNA unwound in electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13) for 22 min at 4°C before electrophoresis was performed for 20 min at 0.74 V / cm and 300 mA. Slides were washed three times in neutralisation buffer (0.4 M Tris, pH 7.5). The samples were fixed in 100% ethanol for 10 min at RT, air-dried and stained with 50 μg/ml propidium iodide. Comets were analysed by fluorescence microscopy using an Olympus BX50 equipped with a ColorView camera (Olympus, Münster, Germany). At least 100 cells were scored in each experiment by means of Comet IV software (Perceptive Instruments Ltd., Bury St Edmunds, UK).

Formalin-fixed and paraffin-embedded samples from 22 primary human BL cases and from tonsils were obtained from the histopathology files of the Pathology Institute, University Wuerzburg. When biopsy material was studied, the permission and consent of patients was received before. Immune stains of paraffin slices were performed with Abs directed against NFATc1 (clone 7A6, BD Pharmingen) and NFATc1/α (anti-IG-457). All pictures were captured with an Olympus Color view camera mounted on an Olympus BX41 dual-head light microscope. The pictures were taken with a Leica Confocal Laser Scanning Microscope (TCS SP5 II) and were analyzed with the Leica Software Image Pro Plus. For further demonstration, the digital images were processed using Adobe Photoshop CS3, Irfan view or Microsoft Office Power Point 2010.
For confocal microscopy, Namalwa and Ramos cells, Eµ-MYC induced mouse primary tumor cells,(#1542T), B cell lymphoma (BCL) cells from the same tumor (#1542B) and naïve splenic B cells were incubated for 1 h with αNFATc1 (7A6), αNFATc1/α (anti-IG-457) and, in some assays, αKi-67. Next, they were stained by secondary fluorescent labelled goat-anti-mouse Alexa Fluor (AF) 488, or goat-anti-rabbit AF 555, or AF 488, or by strepavidin AF 488 (all from eBiosciences), or goat-anti-mouse AF 647 (Dianova), or donkey anti-goat AF 488 (Invitrogen).

Cryostat sections (10 μm thickness) of OCT embedded tumors, were dried on Superfrost Plus slides (Fisher Scientific), fixed with 4% paraformaldehyde and then incubated with 0.2% Triton X-100, 10% normal goat serum to block non-specific antibody binding. Sections were incubated for 15–18 h with primary antibodies at 4°C. Rabbit monoclonal anti-CD3 (AbCam) and rabbit monoclonal anti-CK7 (AbCam) were used. Secondary antibodies were Goat anti rabbit Alexa568 and Alexa488 (Invitrogen), respectively. Tissue sections were examined with an Olympus BX61 fluorescence microscope equipped with single, dual and triple fluorescence filters, and a Colorview camera (Olympus). Digital images of tumors in sequential cryostat sections of each tumor were captured.

Sample processing and whole-mount immunohistochemistry of dissected embryonic lungs at E11.5–E15.5 were performed as described previously (Alanentalo et al., 2007 (link)). Briefly, fixed lungs were dehydrated with methanol followed by rehydration and processing to immunohistochemical staining. Localization of anti-E-cadherin (Cell Signaling Technology) was detected either by fluorescently labeled secondary antibody conjugated to Alexa-Fluor-594-conjugated anti-rabbit IgG (Life Technologies/Thermo Fisher Scientific Inc.) or visualized by using the chromogenic DAB substrate (Immunologic, Duiven, The Netherlands) following the incubation with poly-HRP-conjugated anti-rabbit IgG antibody (Immunologic, Duiven, The Netherlands). Fluorescently labeled lungs were processed for OPT scanning as described previously (Alanentalo et al., 2007 (link)), using a Bioptonics OPT 3001M Scanner. Three-dimensional (3D) visualization and branch end-point analysis was performed with Imaris 3D and 4D data software, using the Filament analysis function (Bitplane AG, Switzerland). Chromogenically stained samples were imaged using a Leica MZFLIII stereomicroscope (Leica, Germany) and Colorview camera (Software imaging system, Olympus, Japan).