Axiovert inverted microscope

Dynamic changes in [Ca²⁺]mito were followed in cells expressing 4mtD3cpv. Medium was removed and cells were kept in loading buffer containing 135 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM Hepes, 2.6 mM NaHCO₃, 440 μM KH₂PO₄, 340 μM Na₂HPO₄, 10 mM D-glucose, 0.1% vitamins, 0.2% essential amino acids, 1% penicillin/streptomycin, pH adjusted to 7.4. Single cell measurements were performed on a Zeiss AxioVert inverted microscope (Zeiss; Göttingen, Germany) equipped with a polychromator illumination system (VisiChrome, Visitron Systems; Puchheim, Germany) and a thermoelectric-cooled CCD camera (Photometrics CoolSNAP HQ, Visitron Systems; Puchheim, Germany). Infected cells were imaged with a 40 × oil-immersion objective (Zeiss). Excitation of the FRET-based genetically encoded Ca²⁺ indicator 4mtD3cpv was at 440 ± 10 nm (440AF21, Omega Optical; Brattleboro, VT), and emissions were recorded at 480 and 535 nm using emission filters (480AF30 and 535AF26, Omega Optical) mounted on a Ludl filterwheel. Devices were controlled and data were acquired by VisiView 2.0.3 (Visitron Systems) software and analyzed with GraphPad Prism version 5.00 for Windows (GraphPad Software; San Diego, CA). Results of FRET measurements are shown as (Ri – Background) + [(Ri – Background) - (R0 - Background)] (where R0 is the basal ratio) to correct for photobleaching and/or photochromism.

The mouse polysialyltransferase ST8SiaIV was subcloned into the pcDNA3 (RRID:Addgene_42208) vector and use as a template for the synthesis of digoxigenin-11-labeled (Roche Molecular Diagnostics, Mannheim, Germany) antisense and sense probes cRNA probe. tg mice and wt littermates were anaesthetized using 2.5% tribromoethanol (Fluka,Germany) and perfused with 4% paraformaldehyde (PFA). Organs were isolated and postfixed, washed and 12 μm frozen sections generated. In situ hybridization on retina, spinal cord and brain sagittal cryosections (12 μm) was done as recently described (Fewou et al. 2005 (link)). Briefly, frozen slice were fixed and hybridization was performed using the appropriate Digoxigenin-11-UTP-labeled antisense or sense cRNA probes at 65°C in 50% formamide, 1% Denhardt’s solution, 0.2% SDS, 0.25 mg/mL ssDNA, 0.25 mg/mL tRNA and 10% hybridization salt (3 M NaCl, 0.1 MPIPES, 0.1 M EDTA) overnight in humid chamber. Unspecific bound were removed by washes in 2X SSC and 0.1X SSC successively. Bound probes were visualized with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Molecular Diagnostics Cat# 11093274910,RRID:AB_514497) and nitro blue tetrazolium/bromo-chloro-indolyl phosphate as substrate. Sections were examined with a Zeiss Axiovert inverted microscope (Carl Zeiss, Jena, Germany).

BAECs transfected with various FRET biosensors or DNA plasmids were starved with 0.5% FBS of Dulbecco's Modified Eagle Medium(DMEM) for 24 hr before 65 dyn/cm² of HSS application. All images were obtained only on isolated single cell by using Zeiss Axiovert inverted microscope equipped with FRET system. Time lapse fluorescence images were acquired and quantified by MetaFluor 6.2 software (Universal Imaging) and the data of FRET efficiency was analysis by Excel (Microsoft).
Additional details are available in the Supplement.