Ec plan neofluar 10 0

Images from immunoperoxidase stained sections were captured using a Leica slide scanner and Zeiss Axioplan 2 microscope; from immunofluorescent stained human sections with a Zeiss Axioimager Z2 apotome; from immunofluorescent stained marmoset sections with an Axio Imager Z1 microscope (Zeiss) equipped with a Zeiss Axiocam HRm digital camera using the Axiovision software (v 4.8.1.0) at a resolution of 1024 × 1024 pixels. The objectives used were Zeiss EC-Plan Neofluar 10×0.3, #420 340–9901. Filter sets used for visualizing fluorescently-labeled cells were Zeiss 49 Dapi #488 049-9901-000, and Zeiss HQ Texas Red #000 000-1114-462. Images were adjusted for brightness and sharpness using Adobe Photoshop CS6 software. Planes of sectioning are illustrated in Supplementary Figure S1.

FRET/cGMP imaging of cells ex vivo was performed in flow chambers (ibidi) using an epifluorescence setup (Supplementary Fig. 3a) based on an inverted Axiovert 200 microscope (Zeiss) equipped with EC Plan NeoFluar 10×/0.3, LD Plan NeoFluar 20×/0.4 air, and Plan NeoFluar 40×/1.3 oil objectives and optional 1.6× Optovar magnification (Zeiss). The imaging setup contains a light source with excitation filter switching device (Oligochrome, TILL Photonics GmbH), a DualView beam splitter with 516 nm dichroic mirror and CFP and YFP emission filters (480/30 nm and 535/40 nm) (Photometrics), and a CCD digital camera (Retiga 2000R, QImaging)^{22 (link)}. Images were acquired at 0.2 Hz or 1 Hz at room temperature. Adherent cells were exposed to flow at a shear rate of 500 s⁻¹ using a syringe pump (B-Braun). Platelet thrombi and VSMCs were superfused at room temperature with platelet Tyrode buffer and imaging buffer (in mM: 5 HEPES, 140 NaCl, 5 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 5 d-glucose, pH 7.4), respectively. Drugs were applied via two sample loops connected in series and controlled by injection valves (Pharmacia V-7, GE Healthcare).

CLSM analysis of defibrillated cellulose samples using Carbotrace 480 was processed using the protocol of Zitzmann et al. [15 (link)]. The samples (20 mg) were mixed and incubated with 50 µL of Carbotrace 480: phosphase buffer solution pH 7.4 (ratio 1:1000) for 30 min. The CLSM images were acquired using a Zeiss LSM980 confocal microscope, AxioObserver Z1, using ZEN 3.4 (blue edition) software and either an EC Plan-Neofluar 10×/0.3 or a Plan Apochromat 20×/0.8 objective. All the samples were excited with a 405 nm laser using a 405 nm main beam splitter, and the emissions were collected from 411 to 694 nm in bins of 8.9 nm.