Neurons transfected with Lifeact-GFP constructs as specified were imaged at 37°C in HBS buffer using a Zeiss Selleckchem Palbociclib LSM510 confocal microscope (Figures 3B–3E and S3C) or a Perkin Elmer Ultraview
spinning disc microscope (Figures 3F, 3G, and S3D). Image conditions were optimized to minimize photobleaching induced by time-lapse imaging. Bleaching was achieved at maximum laser transmission at 488 nm for less than 30 s and targeted to predefined circular regions of interest (ROIs) of approximately 3 μm radius corresponding to individual spines of similar size and morphology. Following bleaching, images were automatically acquired at 5 s intervals, unless otherwise stated. Background fluorescence was subtracted for each frame during the image processing to quantify the recovery. Recovery at time point t was calculated as ROI/REF, where ROI is intensity at the region of interest and REF corresponds to intensity of a “reference” nearby spine to account for minor focus changes during acquisition. Recovery values were normalized to the average intensity of five prebleach frames. Exponential fit to simple regression curves was performed with Sigmaplot software. Values were fit to the equation y = y0 + a(1 − exp(−bx)), where y0, a,
and b are offset, maximum value, and time constant, respectively. To optimize the fit, all curves analyzed were constrained to reach the maximum value of recovery (a + y0), defined as the Megestrol Acetate average of the last three Sunitinib values. The equation t1/2 = ln(0.5)/−b was used to extract half-life of recovery, and conditions were compared using a t test. Organotypic slices were prepared from P8 Wistar rats using the interface method (Bortolotto et al., 2011 and Stoppini et al., 1991). Transverse hippocampal slices (400 μm) were placed on Millicell culture plate inserts (Millipore) and maintained at 35°C, 5% CO2 in MEM-based culture media containing 20% horse serum and
(in mM): 30 HEPES, 16.25 glucose, 5 NaHCO3, 1 CaCl2, 2 MgSO4, 0.68 ascorbic acid, and 1 μg/ml insulin (pH 7.28), 320 mOsm. Biolistic transfection was performed using a Helios GeneGun (Bio-Rad) and electrophysiological recordings were performed, blind with respect to the transfected plasmid (either WT-Arf1-IRES-EGFP or ΔCT-Arf1-IRES-EGFP together with mCherry), 2–4 days later. Whole-cell voltage-clamp recordings were made from CA1 pyramidal cells (Vh = −70 mV) at 6–11 DIV. Patch pipettes contained (in mM) 115 Cs-methanesulfonate, 20 CsCl, 10 HEPES, 2.5 MgCl2, 4 Na2ATP, 0.4 Na3GTP, 10 sodium phosphocreatine, and 0.6 EGTA or alternatively 8 NaCl, 130 Cs-methanesulfonate, 10 HEPES, 0.5 EGTA, 4 MgATP, 0.3 Na3GTP, and 5 QX-314 (pH 7.25, 290 mOsm). Picrotoxin (50–100 μM) and 2-chloroadenosine (1–2 μM) were routinely included in the bath solution (124 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.4 mM NaH2PO4, 4 mM CaCl2, 4 mM MgSO4, 10 mM glucose; saturated with 95% O2/5% CO2).
Related posts:
- Right here we report a fast and sustained phosphorylation of ERK1/2 in neurons i
- Apatinib Ells were transiently transfected with siRNA
- The cells were transfected with siRNA utilizing Lipofectamine RNA
- C-Met Signaling Pathway receptor expression of transfected cDNA construct into pcDNA3
- Chemical compound library empty plasmids were transfected into MCF-7 and MDAMB 231 cells