Acidic intracellular compartments were visualized by acridine orange staining. After seeding, cells were washed with phosphate buffered saline (PBS) and stained with 10 μg/ml acridine orange (Invitrogen, Madison, WI, USA) for 15 min at 37°C. Subsequently, the cells were washed with PBS and viewed under a laser scanning confocal microscope. Microscopic images were collected using the LSM 5 PASCAL software (Carl Zeiss, Jena, Germany). Depending on their acidity, autophagosome or autolysosome appeared as orange or red fluorescent cytoplasmic vesicles, whereas nuclei were stained green. Alternatively, acridine orange-stained cells were trypsinized, washed with PBS, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA) using the CellQuest Pro software (Becton Dickinson).
Lsm 5 pascal software
Manufactured by Zeiss
Sourced in Germany
The LSM 5 PASCAL software is a core component of the LSM 5 PASCAL imaging system developed by Zeiss. It provides the fundamental functionality for controlling and operating the system's hardware and acquiring images. The software enables users to configure and manage the various parameters of the microscope, detectors, and other integrated components to capture high-quality digital images.
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Lab products found in correlation
Facscalibur, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Acridine orange, by Thermo Fisher Scientific (1 mentions)
Acridine orange, by BD (1 mentions)
Hene laser, by Zeiss (1 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Duolink in situ proximity ligation assay pla, by Olink (1 mentions)
Chamber slide, by Thermo Fisher Scientific (1 mentions)
Syto59, by Thermo Fisher Scientific (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
10 protocols using lsm 5 pascal software
1
Acridine Orange Staining of Acidic Organelles
2
Fluorescence in situ Hybridization Analysis of Anammox Bacteria
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Biomass samples were collected from the upflow column reactor and fixed in 4% paraformaldehyde. FISH was subsequently conducted according to the procedure described by Okabe et al. (23 (link)). The 16S rRNA-targeted oligonucleotide probes used in this study were EUBmix, composed of EUB338 (1 (link)), EUB338II, and EUB338III (5 (link)), which are specific for most bacteria; Amx368 (29 (link)), which is specific for all anammox bacteria; and Sca1129a, specific for uncultured bacterium husup-a2, and Sca1129b, specific for the uncultured bacterium husup-a7, which distinguish two phylogenetically different “Ca. Scalindua sp.” in the inoculum used in this study (14 (link)). The probes were labeled with Cy3 or Alexa Flour488 at the 5′ end. A LSM5 PASCAL model confocal laser-scanning microscope, equipped with an Ar ion laser (488 nm) and HeNe laser (543 nm; Carl Zeiss, Oberkochen, Germany), was used for microscopy. The average surface area fraction was determined from at least 24 representative laser scanning microscopy projection images using the LSM5 PASCAL software provided by Carl Zeiss (13 (link)).
3
Mitochondrial Calcium Imaging with Rhod2 AM
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Rhod2 AM was used to determine the mitochondrial Ca2+ level.34 (link) Rhod2 AM has a net positive charge, which facilitates its sequestration into mitochondria via membrane potential-driven uptake. The use of Rhod2 AM improves the selectivity of mitochondrial loading, because the dye exhibits Ca2+-dependent fluorescence only after it is oxidized, a process that occurs preferentially within mitochondria. Cells were seeded at a concentration of 1×105 cells/well; 16 hours after plating, they were treated with DUE at 40 μg/mL and incubated for an additional 48 hours. Cells were harvested, washed, and re-suspended in PBS containing Rhod2 AM. After 15 minutes of incubation at 37°C, cells were washed, suspended in PBS, and analyzed by flow cytometry. For image analysis, cells were loaded with Rhod2 AM and incubated for 30 minutes at 37°C. The stained cells were then washed and mounted on microscope slides in mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images were obtained using a confocal laser scanning microscope and LSM 5 PASCAL software (Carl Zeiss).
4
Immunofluorescence Analysis of Stem Cell Markers
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Confluent RPE and HREC cell monolayers were fixed in ice-cold Acetone/Methanol (1:1) followed by permeabilization in 0.1% Triton X-100. After permeabilization, the monolayers were blocked in 5% BSA in PBS and further incubated with primary antibodies (rabbit polyclonal Prom1, mouse β-catenin, rabbit LC3-I and LC3-II) for 1 hour at 37°C, followed by 1 hour incubation with secondary antibodies (Alexa Fluor 488-conjugated anti-mouse IgG and Cy-3 conjugated anti-rabbit IgG antibodies). For LC3 staining, cells were mounted on glass slides using mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). For localization of Prom1 and β-catenin, the fluorescence was examined under a Zeiss LSM 5 laser scanner confocal microscope, and images were collected using LSM 5 Pascal software as described earlier. Images were stacked using the software, Image J (National Institutes of Health, Bethesda, MD, USA), and processed by Adobe Photoshop (Adobe Systems, Inc., San Jose, CA, USA).
5
Detecting AT1R/μOR Heterodimers in Rat Brain
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The Duolink in situ proximity ligation assay (PLA; OLINK Bioscience, Uppsala, Sweden) was utilized to detect the formation of AT1R/μOR heterodimers. The NTSs of SHRs and WKY were examined to detect the formation of AT1R (sc-515884) and μOR (bs-3623R) heterodimers in situ. The rats were first perfused in saline, then 4% formaldehyde, and finally 30% sucrose solution. Brainstem’s microsections of 5 μm thickness were obtained. Primary antibody diluent (OLINK Bioscience), containing two primary antibodies (1:100 for goat anti-μ receptor antibodies and 1:100 rabbit anti-AT1R antibodies), was added to the sections, and incubated overnight at 4 °C. Next, PLA secondary antibody with specific oligonucleotides, anti-rabbit plus and anti-goat minus (OLINK Bioscience), were applied to the sections and incubated for 2 h at 37 °C. Samples underwent ligation to allow nearby oligonucleotide probe pairs to form closed circles, and signals were amplified in the amplification solution. Images were acquired using a confocal laser scanning microscope (Carl Zeiss LSM 5 PASCAL), and were further processed by LSM 5 PASCAL software (Version 3.5, Carl Zeiss), which automatically counted the number of spots per unit of surface area.
6
Endoplasmic Reticulum Staining and Imaging
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Cells were seeded at a concentration of 1×105 cells/well, and 16 hours after plating, they were treated with DUE at 40 μg/mL and incubated for 48 hours. Cells were harvested, washed, and re-suspended in phosphate buffer saline (PBS) containing the ER-tracker Blue-White DPX probe. After 30 minutes of incubation at 37°C, cells were washed, suspended in PBS, and analyzed by flow cytometry. For confocal imaging analysis, cells were seeded in chamber slides (Nalge Nunc International, Naperville, IL, USA) at a density of 1×105 cells/mL; 16 hours after plating, they were treated with DUE at 40 μg/mL and incubated for 48 hours. Then, ER-tracker was added to the cells, and the samples were incubated for 30 minutes under the same growth conditions. The loading solution was removed, and the cells were washed with PBS before addition of fresh medium without the stain. Microscopic images were collected using the LSM 5 PASCAL software (Carl Zeiss, Jena, Germany).
7
Quantifying Cellular Glutathione Levels
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Cell cultures (1.0×105 cells/mL) were treated with 2.5 μM RA and exposed to UVB 1 h later. Following 24 h of incubation, cells were treated with 10 μM of CMAC dye, and samples were incubated for a further 30 min at 37°C. Additional dye was washed off with PBS, and the stained cells were mounted onto a chamber slide in a mounting medium. The slides were observed under a confocal microscope using the LSM 5 PASCAL software (Carl Zeiss, Jena, Germany). GSH levels were determined by spectrophotometry in either cells or mouse tissues using a BIOXYTECH GSH-400 assay kit (Oxis International Inc., Foster City, CA, USA) following the manufacturer’s protocol.
8
Biofilm Visualization and Analysis Using CLSM
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Different methods have been used to study biofilm formation including CLSM, AFM [19 (link)]. In our study, CLSM has been employed to reveal the biofilm structure as well as the cell viability within biofilms. Biofilms grown on the rough surface of the DBR discs were treated with Polident® experimental formula M138-12 (GlaxoSmithKline, UK) or Efferdent® antibacterial denture cleanser (Prestige brands, USA) for 5 min according to manufacturer’s recommendations, and untreated controls were stained with 10 μM of the cell-permeable fluorescent stain SYTO 59 (labeling of all cells) and 10 μM of the cell-impermeable fluorescent stain SYTOX Green (labeling of cells with compromised cell walls only) (Invitrogen, USA) in PBS buffer for 20 min at room temperature in the dark before visualization [17 (link)]. All biofilm images were collected with a Zeiss LSM 5 PASCAL CLSM using LSM 5 PASCAL software (Zeiss, Germany). Excitation at 633 nm with an argon laser in combination with a 650 nm band-pass emission filter was used for SYTO 59 fluorescence imaging. SYTOX Green signals were visualized using 488 nm excitation with a helium-neon laser and a 503-530 nm band-pass emission filter.
9
Cytoskeleton Protein Analysis in Cultured Cells
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Production and arrangement of cytoskeleton proteins were studied during the entire experimental period. Briefly, the cells were detached using trypsin/EDTA (0.25%; Thermo Fisher Scientific), counted using a hemocytometer, and seeded at a density of 10,000 cells/cm2 in gelatin (0.1%; Thermo Fisher Scientific)-coated 12-well plates (Corning) at a density of 10,000 cells/cm2. The day after, the cells were stained for F-actin and the type III intermediate filament protein vimentin. Upon reaching optimal density for imaging, the cells were fixed using a 4% paraformaldehyde solution in DPBS (v/v) (Chem Cruz®, Santa Cruz) as described above. Thereafter, the cells were stained with Alexa Fluor® 488 Phalloidin (1:40 in blocking solution; Thermo Fisher Scientific, cat. no. A12379) and vimentin monoclonal antibody (V9) eFluor 615 (1:500 in blocking solution; Thermo Fisher Scientific, cat. no. 42989782). The cell nuclei were counterstained with DAPI (1:1000 dilution) (BioVision). Confocal images were taken with the microscope Axiovert 200 M microscope (Zeiss, Oberkochen, GE) mounted with LSM 5 Pascal exciter using the LSM 5 Pascal software (Zeiss).
10
Quantifying Cellular Glutathione via Confocal Microscopy
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For image analysis of GSH, cells were seeded in four-well chamber slides at a density of 1 × 105 cells/mL. Sixteen hours after plating, cells were treated with 20 μM fucoxanthin and then irradiated with UVB 1 h later. After 12 h, 10 μM of CMAC was added to each well, and samples were incubated for an additional 30 min at 37 °C. After washing with PBS, the stained cells were mounted onto a chamber slide in mounting medium. Images were collected on a confocal microscope using the LSM 5 PASCAL software (Carl Zeiss, Jena, Germany). In addition, the GSH concentration was measured using a BIOXYTECH GSH-400 assay kit (Foster City, CA, USA).
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