Tcs sp2 confocal microscopy

To measure intracellular Ca²⁺, cells were loaded with 5 μM Fluo-4 AM (ThermoFisher Scientific, Waltham, MA) for 20 minutes and washed three times and maintained in the following solution (in mM): NaCl 125, KCl 4.75, MgSO₄ 1.2, KH₂PO₄ 1.2, HEPES 30, glucose 10, taurine 50, CaCl₂ 2 and pH = 7.4. A Leica TCS SP2 confocal microscopy with 40x, 1.25 NA oil immersion objectives was used for linescan imaging. The scan zoom was adjusted to fit the cells, and scan line was performed along the long axis of cells49 . The excitation for Fluo-4 was 488 nm, while emission was collected at 505–530 nm. For Ca²⁺ sparks recording, cells were scanned at 400 Hz for 20 s following 1 minute of pacing at 3 Hz. Ca²⁺ sparks detection and analysis was performed as previously described49 50 (link)51 (link). KN-93 was purchased from Sigma-Aldrich (St. Louis, MO). For simultaneous recording of Ca²⁺ transient amplitudes and SR Ca²⁺ contents, cells were exposed to 10 mM caffeine immediately following termination of pacing at 1 Hz for 1 minute. Sampling started 10 s before caffeine treatment.

Mouse ECs transduced with lentiviral vectors carrying shRNA for Nova2 were seeded in 35-mm Petri dishes coated with Gelatin (Difco) 0.1% and cultured for 72 h. The splitting ratio was such that confluence was reached overnight after seeding. For immunofluorescence, ECs were fixed with 4% paraformaldehyde (PFA) and then permeabilized with 0.5% Triton X-100 for 10 min. Blocking (1 h), primary (overnight) and secondary (1 h) antibodies were diluted in PBS with 2% BSA. The following primary antibodies were used: anti-Par3 (1:100 Millipore), anti-Podocalyxin (1:100 R&D), anti-VE-cadherin (1:100 C-19, sc-6458, goat, Santa Cruz Biotechnology) and anti-β-catenin (1:50 BD Transduction Laboratories). Secondary antibodies for immunofluorescence were donkey antibodies to the appropriate species conjugated with Alexa Fluor 488, 555 or 647 (dilution 1:200 or 1:400).
For imaging, charge-coupled device camera on epifluorescence microscope (Leica) or Leica TCS SP2 confocal microscopy were used. ImageJ (NIH) was employed for data analysis. Figures were assembled using Adobe Photoshop and Adobe Illustrator. Only adjustments of brightness and contrast were used in the preparation of the figures. For comparison purposes, different sample images of the same antigen were acquired under constant acquisition settings.

DNA regions encoding the N-terminal targeting sequence of each MISF protein were PCR amplified, cloned into pDONR207 by Gateway^TM BP reaction (Invitrogen) and sequenced to check PCR accuracy. The obtained entry clones were subsequently transferred into pGWB5 (Nakagawa et al., 2007 (link)) by the Gateway^TM LR reaction (Invitrogen) to create a translational fusion between the targeting sequences and the coding region of green fluorescent protein (GFP). The fusion constructs were transformed into Agrobacterium tumefaciens C58C51 and used for floral dip transformation of Arabidopsis Col-0 plants. Transgenic plants were selected on hygromycin, and GFP fluorescence was visualized in root cells by Leica TCS SP2 confocal microscopy. Prior to observation, roots were soaked in a solution of Mitotracker^TM Red (0.1 μM) to label mitochondria.