Neonatal rat ventricular cardiomyocytes (NRVMs) were plated on glass coverslips coated with poly-D-lysine in 12-well plates. Cardiomyocytes were fixed with 2% paraformaldehyde for 20 min with 2% paraformaldehyde. Immunofluorescence was performed as previously described [27 (link)]. Primary antibodies employed include mouse anti-Troponin-T (1:200; Abcam, Cambridge, UK; ab8295), rabbit anti-CaVα2δ1 (1:200, Alomone, Jerusalem, Israel, ACC-015), and rabbit anti-CaVα1C (1:200, Alomone, ACC-003). Secondary antibodies employed include goat anti-mouse coupled to Alexa 555 (1:800; Invitrogen, Waltham, MA, USA; A21424) and donkey anti-rabbit coupled to Alexa 488 (1:800; Invitrogen; A21206). The nucleus was identified with 4′,6′-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) staining, and 4′,6-diamidino-2-phenylindole DAPI (1:800). Images were captured by confocal microscope with 20× or 63× objective. Images were analyzed using Zeiss LSM image software. The cross-sectional area (µm2) of 250 Troponin-T+ mononucleated cardiomyocytes from 5 different images per condition was determined using Zeiss LSM image software. For the colocalization analysis, wheat germ agglutinin (WGA) antibody (1:200; Invitrogen; W32466) was added to cardiomyocytes prior to fixation.
Lsm image software
Manufactured by Zeiss
Sourced in Germany
The LSM image software is a powerful tool designed for image data acquisition, processing, and analysis. It provides a comprehensive set of features for users to capture, visualize, and analyze high-quality images from various microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 or 588 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Shandon cryomatrix, by Thermo Fisher Scientific (3 mentions)
Lsm 710 meta inverted confocal microscope, by Zeiss (3 mentions)
Zen desk software, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Dihydroethidium, by Thermo Fisher Scientific (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Dapi fluoromount g, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Ab15106, by Abcam (1 mentions)
Ab34269, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
E 600me microscope, by Nikon (1 mentions)
Mca1031g, by Bio-Rad (1 mentions)
Alexa fluor 488 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p fak, by Cell Signaling Technology (1 mentions)
Rabbit anti p src, by Cell Signaling Technology (1 mentions)
Rabbit anti actin, by GeneTex (1 mentions)
29 protocols using lsm image software
1
Immunofluorescence Analysis of Neonatal Rat Ventricular Cardiomyocytes
2
Liposomal Drug Delivery and Tumor Hyperthermia
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Liposome binding to tumor vasculature and their clearance from circulation was analysed by intravital microscopy after injection of DiD-labelled TSL or RGD-TSL and followed up to 24 or 5 h respectively. In order to evaluate Dox release during hyperthermia and its uptake by tumor vascular endothelial cells and tumor cells, DiD-labelled Dox-TSL or RGD-Dox-TSL were injected i.v. through the penile or tail vein at a dose of 5 mg/kgDox. Both formulations were allowed to circulate for 5 h at body temperature in order to be able to bind to vascular angiogenic endothelial cells or tumor cells and observed by confocal microscopy (Zeiss LSM 510 META). After 5 h of circulation, tumor was heated at 42°C for 1 h and Dox release and uptake was detected as above (20× objective lens). Regions of interest were selected before, during and in the end of the hyperthermia treatment. Images of 1024 × 1024 pixels were analyzed using Zeiss LSM image software (Zeiss, Germany).
3
Immunofluorescence Analysis of Oxidative Stress Markers
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Cryostat slices (10 μm) or the lens capsular flaps were incubated with anti-NOX4, anti-GLUT1, anti-GLUT5, anti-RAC1 or anti-RAGE antibodies (1:100). After being washed with phosphate-buffered saline, sections were incubated with green-fluorescent Alexa Fluor 488- or 588-conjugated donkey anti-rabbit IgG (1:200; Invitrogen) at 25 °C for 2 h. The sections were analyzed using fluorescence microscopy and Zeiss LSM Image software (Carl Zeiss MicroImaging).
4
Immunohistochemical Profiling of Brain Injury
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Immunohistochemistry was performed as previously described54 (link), 55 (link). Free floating, fixed brain sections with 40-μm thickness underwent fluorescent immunohistochemical staining using the following primary antibodies: anti-glial acidic fibrillary protein (GFAP) (OPA1-06100, Thermo Fisher, 1:1000 dilution), anti-CD45 (MCA1031G, BioRad, 1:500 dilution), anti-mouse fibrinogen (ab34269, Abcam, Cambridge, UK, 1:1000 dilution), and anti-claudin 5 (ab15106, Abcam, Cambridge, UK, 1:500 dilution). The secondary antibodies used included Alexa Fluor 488 Goat Anti-Rabbit IgG (A-11008), Alexa Fluor 488 Goat Anti-rat IgG (A-11006), and Alexa Fluor 350 Goat Anti-rat IgG (A-21093, Invitrogen; 1:500 dilution). Stained sections were imaged using a Zeiss LSM5 confocal scanning microscope system and analyzed using the Zeiss LSM Image software (Zeiss Ltd., Jena, Germany). Another cohort of brain sections underwent 3,3′-Diaminobenzidine (DAB) staining with anti-mouse fibrinogen (ab34269, Abcam, Cambridge, UK, 1:3000 dilution). Stained sections were imaged using a Nikon E-600ME microscope and analyzed using MetaMorph software. Immunohistochemical images were taken of the left cerebral cortex (ipsilateral to the lesion).
5
Dasatinib Alters E-Cadherin and Vesicle Trafficking in HT-29 Cells
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HT-29 cells were grown on coverslips for 24 h and treated with 20 nM dasatinib for 24 h. The cells were fixed with 3.7 % formaldehyde for 20 min and permeabilized with 0.1 % Triton X-100 for 1 min. The fixed cells were incubated with mouse anti-E-cadherin (BD Biosciences), rabbit anti-p-Src (Cell Signaling Technology), rabbit anti-actin (GeneTex), rabbit anti-Rab11 (Invitrogen), rabbit anti-pEGFR (Invitrogen) and rabbit anti-p-FAK (Cell Signaling Technology) for 1 h at room temperature, followed by incubation with secondary antibodies conjugated to Alexa Fluor 488 or 594 (Jackson ImmunoResearch) for 1 h at room temperature. After washing with PBS, the coverslips were mounted with Fluoromount (Merck-Sigma, MA, USA), and images were acquired using a Zeiss LSM 510 META confocal system with a 63X objective (1.4 oil). Z-scanning sections were taken for 3D imaging, and line scans and side views were analyzed using Zeiss LSM image software. For quantification of E-cadherin, Rab11 and pEGFR distribution at leading edge and the cell-cell contacts, more than 100 cells of each independent experiment were counted, the positive counts were divided by the total cell-cell contact numbers.
6
Cellular Uptake and Dox Release Assay
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B16Bl6, B16F10 or HUVEC cells were seeded at the same concentrations as for fluorescent microscopy in cell culture chambers containing a cover glass insert coated with 0.1% gelatine. Cells were allowed to recover for 24 h. After 24 h, cells were incubated with 400 nmol/ml NBD-PE labelled TSL or RGD-TSL for 3 h at 37°C and for 30 min with lysotracker (LysoTracker® Red DND-99). After incubation, cells were washed three times with DMEM (B16Bl6 and B16F10) or HUVEC medium (HUVEC). Cells were analyzed on a Zeiss LSM 510 META confocal laser scanning microscope. NBD-PE fluorescence was detected by 513 nm argon laser and lysotracker was monitored by a 543 nm Helium –Neon laser. For Dox release experiments, cells were incubated with NBD-PE or DiD labelled Dox-TSL or RGD-Dox-TSL for 3 h at 37°C and after that washed three times with medium with FCS. Dox release was followed in time for 1 h at 42°C (40 × objective lens, 2,5 μm pinhole) and its fluorescence was detected by a 543 nm Helium –Neon laser. Images of 1024 × 1024 pixels were analyzed using Zeiss LSM image software (Zeiss, Germany).
7
Immunostaining of Cryostat Slices
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Cryostat slices (10 μm) or the lens capsular flaps were incubated in anti-NOX4, anti-GLUT1, anti-GLUT5, anti-RAC1 or anti-RAGE primary antibodies (1:100 dilution). After a phosphate-buffered saline wash, sections were incubated in green-fluorescent Alexa Fluor 488- or 588-conjugated donkey anti-rabbit IgG (1:200 dilution; Invitrogen) at 25 °C for 2 h. The sections were analyzed using a fluorescence microscope and Zeiss LSM Image software (Carl Zeiss MicroImaging, DE Thüringen, Jena, Germany).
8
Immunofluorescence Staining Protocol
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The samples were permeabilized with 0.5% Triton X-100 for 15 min, then blocked with 10% normal horse serum for 30 min. Afterward, the specimens were incubated with different primary antibodies in 10% normal horse serum at 4℃ overnight. Specimens were then treated with secondary antibodies in 10% normal horse serum for 2 h at 24–26 °C. Finally, the sections were mounted and counterstained with mounting medium which contains DAPI. The sections were analyzed using fluorescence microscopy and Zeiss LSM Image software (Carl Zeiss MicroImaging, DE Thüringen, Jena, Germany).
9
Immunofluorescent Analysis of Brainstem
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The rats were perfused using 0.9% saline and 4% formaldehyde, followed by 30% sucrose solution. Brainstems were cut into 20 μm-thick sections, incubated in anti-endomorphin-2, anti-IBA1, anti-AT1R (ab124505) and anti-nNOSS1416 primary antibodies at a dilution ratio of 1:100. After washing with PBS, sections were incubated in Alexa Fluor 488 or 588-conjugated donkey anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA, USA) at 25 °C for 2 h, and analyzed using a fluorescence microscope and Zeiss LSM Image software (Carl Zeiss MicroImaging).
10
In Vivo Lens Oxidative Stress Levels
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The endogenous in vivo O2¯ levels produced in humans with DM cataracts and fructose-fed rats were determined by staining the anterior region of the lens capsule with dihydroethidium (DHE; Invitrogen, Carlsbad, CA). Lens epithelial cell (LEC)-containing slices removed from the rats were placed in OCT compound (Shandon Cryomatrix; Thermo Electron Co., Pittsburgh, PA), flash-frozen in a methylbutane-chilled bath, and then placed in liquid nitrogen. Lens capsular flaps were stained with 1 μM DHE in the dark for 20 min at 37 °C in a humidified 5% CO2 incubator. The samples were analyzed using fluorescence microscopy and the Zeiss LSM Image software (Carl Zeiss MicroImaging, Jena, Germany).
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