Lsm zen software

For FRAP measurements, a 63× 1.4-NA Plan-Apochromat objective was used on an inverted LSM800 system. Photobleaching of GFP was performed using four to six rapid scans with the laser at full power. Pre- and post-bleach images were captured at 0.5- to 3-s intervals, using low laser intensity. Fluorescence recovery in the bleached region during the time series was quantified using LSM Zen software (Carl Zeiss MicroImaging). For presentation purposes, 16-bit confocal images were exported in TIFF, and their contrast and brightness were optimized in Adobe Photoshop software or ImageJ. The characteristic fluorescence recovery time (τ) values for membrane-bound Sec24b turnover were calculated from the photobleaching data by fitting the data to a simple exponential equation using Kalaidagraph software (Synergy Software), $y=Mf · \left(1-e^{-kt}\right),$ where Mf is mobile fraction. R² = 0.98 and 0.97 for WT and mutant, respectively.

To assess biofilm formation, microscopic observations and image acquisitions were performed by CLSM as reported by Sanchez et al.¹⁷ We used a Zeiss LSM 780 (Carl Zeiss Co., Ltd, Oberkochen, Germany) on days 3, 5, 7 and 14 after bacterial inoculation into the samples, which were sufficiently long for the biofilm to mature.¹⁹ 3D images and optical z‐sections were generated using LSM ZEN‐software (Carl Zeiss Co., Ltd). Three image stacks were acquired from each sample at 1‐μm intervals in z‐section from the substratum to the top of the biofilm. Three independent experiments were performed according to the same protocol described above. In total, nine image stacks were acquired and analyzed for each DLC‐coated/uncoated sample.

DNA fiber assays were performed following published protocols (56 (link), 57 (link)). Briefly, PC-3 or U2OS cells were infected with lentivirus expressing EV or CBX3. The cells were first labeled with 25 mM IdU (Cayman, catalog no. 20222-1) for 30 min, washed three times with PBS, and then labeled with 250 mM CldU (Cayman, catalog no. 18155-50) for 1 hour. The labeled cells were harvested, and DNA fibers were spread by gravity. The primary antibodies and dilutions used were mouse anti–5-bromo-2′-deoxyuridine (BrdU) at 1:20 (for IdU) and rat anti-BrdU (for CldU) at 1:100. Images of well-spread DNA fibers were acquired using an LSM700 confocal microscope with a 100× oil immersion objective (Carl Zeiss). Analysis of double-labeled replication forks was performed manually using LSM ZEN software (Carl Zeiss).

To assess the effect of drug/vehicle treatment on newly generated hippocampal neurons, at 24 hours after the last URB597 injection rats were transcardially perfused. Their brains were removed from the skull, post-fixed overnight at 4°C, and transferred first into a 15% sucrose solution, then 24 hours later into a 30% sucrose solution in phosphate-buffered saline. Coronal brain sections (40 μm thick) were then cut and prepared for immunofluorescence analysis with an anti-DCX antibody (1:200; Cell Signaling Technology Inc., Danvers, MA). As previously described (Meneghini et al., 2014 (link)), 15 DCX⁺ immunopositive cells (5 each in 3 different brain coronal sections) located in the DG blades and bifurcation were randomly chosen and their apical dendrite traced throughout its entire extension at 40x magnification using the Zeiss LSM Zen software. The number of DCX⁺ neuroblasts/mm³ in the section samples was evaluated using the same software.

Microscope images were acquired using a confocal microscope (LSM510; Carl Zeiss) mounted on inverted microscope (Axiovert 100M; Carl Zeiss) equipped with a Plan-Apochromat 63×/1.4 oil immersion objective (Carl Zeiss) at RT. Image acquisition and analysis was performed using LSM ZEN software (Carl Zeiss). Dual-color confocal images were acquired using laser lines 488 and 543 nm for excitation of Alexa Fluor 488 and 568 dyes (Invitrogen), respectively. Automated multichannel image acquisition was performed using high-content screening station scan^R (Olympus) equipped with motorized microscope (IX81; Olympus), UPlanSApo 40×/0.95 air immersion objective (Olympus), and digital monochrome electron multiplying charge coupled device camera (C9100; Hamamatsu). Image acquisition and analysis was performed using scan^R acquisition and analysis software (Olympus).