Dm irbe microscope

Indirect immunofluorescence was performed as previously described [5 (link)] by using the anti-EB1 antibody and anti-mouse antibody Alexa 568 nm (Molecular Probes); and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich). Cells were observed using a Leica DM-IRBE microscope, 100X magnification. All images were acquired using Metamoph software (Molecular Devices, Sunnyvale, CA) at identical acquisition settings, and were processed using Image J software. After cell lysis, 30 μg of total protein were loaded into a 12% SDS-PAGE gel. Anti-EB1 antibody, anti-α-tubulin and anti-mouse IgG-horseradish peroxidase (Jackson Immunoresearch) were used. Chemiluminescence detection kit (Millipore) was used for visualization of protein bands. Chemiluminescent signal was acquired on a G:BOX imaging system (Syngene, Cambridge, UK) and quantification was done with Image J software.

Cells were imaged at 20 × magnification using a Hamamatsu Orca 1-megapixel CCD camera fitted to a Leica DMIRBE microscope. Images were captured and analyzed using Volocity (Improvision). Four fields were selected at consistent areas of the peri-lesional region corresponding to each corner of the injury site in each of the sections using standard DAPI, fluorescein, and Rhodamine filter sets. To quantify the cell numbers, a 200-μm × 200-μm (40,000 μm²) square grid was placed at random within each of the four fields around the peri-lesional area. Cell counts were obtained in each area for Hoechst, EdU, GFAP, and double-labeled cells per 40,000-μm² grid. For three brains in each group at least three sections containing the injury site were analyzed. All images for presentation were taken with a Leica SP5 confocal microscope.

The cytotoxicity assay was measured by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. It is based on the reduction of yellow MTT by mitochondrial succinate dehydrogenase. MTT enters the live cells and reduced into insoluble formazan complex. For this, QBC939 cells were seeded at a density of 1 × 10⁴ in a 96-well plate. After 24 h, cells were exposed to blank polymer, free DTX and CSH-DTX at different dosing level. The cells were incubated for 24, 48 and 72 h accordingly. At each time point, plate was removed and treated with 100 μl of MTT solution (5 mg/ml) to each 96-well plate and incubated for 4 h. The formed formazan crystals were extracted by adding DMSO and incubated for additional 30 min. The absorbance of each plate was read at 570 nm using a microplate reader (Thermo-Fisher, USA). All experiments were repeated 6 times.
The morphology of cells was observed using a fluorescent microscope (Leica DM IRBE microscope) and representative images were selected.

For the physiological analysis, female flies were collected just after eclosion and were maintained for 7 days at 29°C. For the heart beat recordings, semi-intact heart preparations were made as previously described (Ocorr et al., 2007 (link); Magny et al., 2013 (link)). An inverted Leica DM Irbe microscope, connected to a DFC450C Leica digital camera, was used to take 20 s recordings at 29 frames/s. Different cardiac parameters were measured using Fly_heart_analysis (SOHA) software based on Matlab R2009b (MathWorks, Natick, MA, USA) (Ocorr et al., 2007 (link)). For the statistical analysis, Student’s t-test was used with Welch's correction when the variances were different.

MEFs were seeded on poly-l-lysine-coated coverslips, serum starved for 22 hr and then left unstimulated or were stimulated with 10% FCS, 2 μM cytochalasin D or 10 μg/ml anisomycin for 30 min. Where indicated, 1 μM BIRB was added to the cells 30 min prior to stimulation. For low calcium treatment cells were starved for 6 hr, washed once with PBS and then incubated in LCM for the time indicated. After treatment cells were washed once with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilised with 0.2% Triton for 5 min. After 1 hr of incubation with 10% heat inactivated Horse Serum (iHS) in PBS, cells were incubated for 1 h with anti-MRTF-A (C-19, sc-21558) primary antibodies (1:200 diluted with 5% iHS/PBS). After three PBS washes, secondary antibodies - Cy3 donkey anti-goat (705–165–003 Jackson ImmunoResearch) together with Phalloidin (Alexa Fluor 647 A-22287) antibodies (1:500 diluted with 5% iHS/PBS) were added and incubated for 1 hr. After 0,5 μg/ml DAPI (ROTH) staining for 5 min coverslips were three times washed with PBS and then mounted with a help of Roti-Mount Fluor Care (ROTH) and analysed using a Leica DM IRBE microscope (40× oil immersion objective) with the Leica TCS confocal systems program.

For indirect IF, cells were cultured on pretreated or coated-glass coverslips (12 mm) and processed as previously described [35 (link)]. Both primary and secondary antibodies were diluted in PBS (pH 7.4) containing 5% BLOTTO or 2% BSA. For some experiments, nuclei were counterstained with 10 ng/ml 4′,6-diamidino-2-phenylindole (DAPI)-PBS (pH 7.4) and samples were mounted in glycerol-PBS (9:1) containing 0.1% phenylenediamine. IF signals were viewed with a DM RXA microscope (Leica, St-Laurent, QC, Canada) equipped for epifluorescence and digital imaging (MicroMax 1300YHS cooled CCD; Princeton Instruments, Trenton, NJ). Numerical images were processed using the MetaMorph Imaging System (Universal Imaging Corp., West Chester, PA) and the Adobe® Photoshop® software (Adobe, San Jose, CA). Cell spreading areas were measured numerically using the MetaMorph Imaging System (Universal Imaging Corp.). For optical inverted contrast microscopy, cells were cultured on plastic culture dishes and viewed with a DM IRBE microscope (Leica) equipped for digital imaging (Princeton Instruments).