Bx51wi upright microscope

Primary cortical neuronal cultures were prepared from embryonic day 17 (E17) Sprague Dawley rats (Envigo and Charles River Laboratories). Neurons were plated at 450,000 cells/well in 12-well plates. On day in vitro (DIV) 12 the neurons were transfected with CACNB4/Myc (OriGene; Catalog #: RR204310) and GFP (gift of Ryan Logan, University of Pittsburgh, PA). Thus, in the same plate some neurons were transfected with both constructs and some with only one. Neurons were fixed on DIV 15 for imaging with mouse anti-c-Myc antibody (1:1000, monoclonal 9E10; Santa Cruz Biotechnology, Inc.) or with mouse anti-CACNB4 (1:100, monoclonal S10-7; antibodies-online Inc.), and goat anti-c-Myc (1:100, polyclonal; Novus Biologicals). Image acquisition was performed on an Olympus (Center Valley, PA) BX51 WI upright microscope equipped with an Olympus spinning disk confocal (SDCM) using an Olympus PlanAPO N 10X 0.40 NA air objective and a 1.42 numerical aperture 60X oil supercorrected objective.
Neurons were first categorized as either CACNB4-overexpressing or GFP-only controls based on c-Myc intensity. GFP-positive neurons on coverslips stained for both c-Myc and CACNB4 were imaged at 10x. Exposure times for the 488 channel were optimized whereas the 568 and 647 channels were shot at fixed exposures of 447 ms and 3000 ms, respectively.

An Olympus BX51WI upright microscope and a water-immersion LUMPlan FL/IR 20×/0.50 W objective were used. Excitation (740 nm) was provided by a Prairie View Ultima multiphoton laser scan unit powered by a Millennia Prime 10 W diode laser source pumping a Tsunami Ti: sapphire laser (Spectra-Physics, Mountain View, CA, USA). Blood plasma was labeled by i.v. tetramethylrhodamine isothiocyanate dextran (155 kDa) in physiological saline (5 % wt/ vol). All microvessels in an imaging volume (500 × 500 × 300 μm) were scanned at each study point, measuring the diameter and blood flow velocity in each vessel (3–20 μm Ø). Tetramethylrhodamine fluorescence was band pass filtered at 560–600 nm and NADH autofluorescence at 425–475 nm. Imaging data processing and analysis were carried out using the NIH ImageJ.

Briefly, Swiss CD-1 mice (postnatal day 14–24) were anesthetized by 3.5% isoflurane inhalation, followed by decapitation. Whole brains were harvested quickly and immersed in ice-cold slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 1.25 NaH₂PO₄-H₂O, 25 NaHCO₃, 0.5 CaCl₂, 7 MgCl₂-6H₂O, 25 d-glucose, 11.6 sodium ascorbate, and 3.1 sodium pyruvate, equilibrated with 95% O₂-5% CO₂ (pH 7.4, 310 ± 5 mosmol/kg). Sagittal slices (300 μm) containing hippocampus were cut with a Dosaka EM DTK-1000 vibratome (Kyoto, Japan) and transferred to an incubating chamber. Slices were then incubated for 15 min at 34°C in an incubating solution containing (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄-H₂O, 25 NaHCO₃, 0.5 CaCl₂, 3.5 MgCl₂-6H₂O, 25 d-glucose, 4 sodium lactate, 2 sodium pyruvate, and 0.4 ascorbic acid (pH 7.3, 310 ± 5 mosmol/kg) before being transferred to room temperature. Slices were then individually transferred to a recording chamber (room temperature) fixed to the stage of an Olympus BX51WI upright microscope fitted with a 40× water-immersion objective lens (0.8 NA). All animal procedures were conducted using protocols approved by the University of Connecticut Institutional Animal Care and Use Committee.