Axiocam mr digital camera

Endothelial-cell spheroids were generated as described previously (36 (link)–39 (link)). Briefly, HUVEC were suspended in culture medium containing 0.2% (w/v) carboxymethylcellulose (Sigma-Aldrich) and seeded in round-bottom 96-well plates (Greiner, Germany) to form spheroids. When siRNA transfection was performed, spheroids were formed 24 h after the transfection of HUVEC with Del-1 siRNA or control siRNA. The following day, spheroids were embedded into rat collagen I (BD Biosciences, Germany) containing gels; after gel polymerization, cells were treated with bFGF-containing medium (bFGF, 30 ng/mL; PeproTech, Hamburg, Germany). After 24 h, images were acquired using an Axiocam MR digital camera with an Axiovert 100M inverted microscope using as objective a Plan-NEOFLUAR (at 10x/0.30) and were processed using AxioVision Rel 4.5 digital imaging software (all from Carl Zeiss, Jena, Germany). In vitro capillary sprouting was quantified by measuring the cumulative sprout length of each spheroid using a computer-assisted microscope (AxioVision 4.5 software, Carl Zeiss) and the mean cumulative sprout length of 10 spheroids/condition was calculated.

HEK (human embryonic kidney)-293T cells were purchased from the A.T.C.C. (Manassas, VA, U.S.A.) and maintained in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (Gibco) supplemented with 10 % FBS. Cells (5 × 10⁵) were seeded in each well of a six-well plate in 2 ml of medium. Inhibitors were added to each well at a final concentration of 0.2, 1, 5, 10 or 15 μM in the presence of 1 % DMSO. For the control sample only 1 % DMSO was added to the well. Cells were trypsinized after 48 h and deposited on to microscope slides by cytospin (200 rev./min for 10 min). For GFP visualization, cells were transfected using Lipofectamine^™ 2000 according to the manufacturer’s protocol (Invitrogen). Transfected HEK-293T cells were FACS-sorted on the basis of GFP intensity (BD FACSAria II). Cytospinning, fixation and immunofluorescence were performed as described previously [27 (link)]. The primary antibodies used for immunofluorescence include anti-H3K9me3 (catalogue number 07-523; Millipore), anti-H4K20me3 (histone H4 trimethylated at Lys²⁰; catalogue number 39180; Active Motif) and anti-HP1γ (catalogue number MAB3450; Millipore). Images were collected on a Zeiss Axiovert 200 imaging system equipped with an Axiocam MR digital camera controlled by AxioVision software or on a Zeiss LSM Pascal confocal microscope.

Mice were anesthetized by intraperitoneal injection of Avertin (400 mg/kg) prior to transcardial perfusion with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. The brains were removed, post-fixed overnight in 4% PFA, and sectioned in the coronal plane at 40 μm thickness. To obtain tangential sections of sensory cortices, the neocortical sheets were flattened between two glass slides, post-fixed, and sectioned tangential to the pial surface at 50 μm thickness. Free-floating sections were used for immunostaining with the following antibodies: rabbit α-5-HT (1:50, Dr. W.M. Steinbusch, University of Limburg, the Netherlands), mouse α-Satb2 (1:1000, Abcam), rabbit α-SERT (1:1000, Millipore), and guinea pig α-Vglut2 (1:2000, Millipore). Immunostaining of the proteins and 5-HT was visualized by using Alexa Fluor 488 or 568 conjugated secondary antibodies. Sections were counterstained with DAPI to detect cell nuclei. Fluorescence images were captured using an Axiocam MR digital camera attached to an AxioImager Z1 microscope (Zeiss). For all the experiments, SERT mutant and control littermates were always analyzed in parallel.