To visualize the dynamics of VP4 molecules in living cells, DF-1 cells growing on glass-bottomed dishes (MatTek, Ashland, MA) were transfected with the recombinant plasmids pEGFP-wtVP4 or pEGFP-VP4-CΔ2 using Lipofectamine 2000 (Invitrogen). At 8 h following transfection, the nuclei of living cells were stained with Hoechst 33342 (Sigma) for 20 min. Cells were then rinsed with PBS twice and supplied with fresh medium. The expression dynamics of EGFP-wtVP4 and EGFP-VP4-CΔ2 were visualized using a Zeiss LSM 700 laser scanning microscope in a humidified cell culture chamber with 5% CO2 at 37 °C.
Lsm 700 laser scanning microscope
Manufactured by Zeiss
Sourced in Germany, United States
The LSM 700 Laser Scanning Microscope is a high-performance optical microscope designed for advanced imaging applications. It utilizes a laser as the light source and a scanning mechanism to capture detailed images of specimens. The LSM 700 is capable of generating high-resolution, three-dimensional images of samples.
Automatically generated - may contain errors
Lab products found in correlation
Zen 2009 imaging software, by Zeiss (19 mentions)
Rpmi 1640 defined medium, by Merck Group (18 mentions)
Syto17 dye, by Thermo Fisher Scientific (18 mentions)
Zen software, by Zeiss (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Alexa fluor 488 goat anti guinea pig, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Hoechst dna staining dye, by Merck Group (2 mentions)
35 mm glass bottomed culture dishes, by MatTek (2 mentions)
Zen 2011, by Zeiss (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Poly lysine coated glass cover slips, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Ssea 4, by Merck Group (2 mentions)
Ssea 1, by Merck Group (2 mentions)
Nanog, by Merck Group (2 mentions)
Zen 2012 light edition program, by Zeiss (2 mentions)
Triton x 100, by Merck Group (2 mentions)
99 protocols using lsm 700 laser scanning microscope
1
Visualizing VP4 dynamics in living cells
2
Immunohistochemical Staining Protocol
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Tissue dissections were performed in PBS followed by fixation in 4% PFA for 20 minutes at room temperature. For this study, the following primary antibodies were: mouse anti-FLAG M5 (1:1,000) (Sigma); rabbit anti-Phm (1:200) [30 (link)], guinea pig anti-Spok (1:200) [61 (link)]; guinea pig anti-Sro (1:1,000) [62 (link)]. Tissues were incubated over night with primary antibodies at 4°C. Fluorescent conjugated secondary antibodies used in this study, goat anti-mouse Alexa Fluor 488, goat anti-guinea pig Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555 and goat anti-guinea pig Alexa Fluor 555, were purchased from Life Technologies. Secondary antibodies were diluted 1:500 and incubated for 1 hour at room temperature. Confocal images were captured using Carl Zeiss LSM 700 laser scanning microscope.
3
Cardiac Fibrosis and Apoptosis Analysis
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Paraffin-embedded heart tissue slices were fixed with 10% neutral buffered formalin and washed with PBS. For immunofluorescent staining of active caspase-3, fixed sections were incubated with anti-cleaved caspase-3 primary antibodies (1:200 dilution, Abcam cat. no. ab49822) overnight at 4 °C. Following three washes with PBS–Tween each at 5 min, the slides for cleaved caspase-3 staining were incubated with Alexa Fluor 488 goat anti-rabbit IgG (A11001; Invitrogen) secondary antibodies (1:200) for 2 h. Then, the slides were washed (5 × 5 min), and co-staining of DAPI was performed to identify tissue nuclei. Assessment of fluorescent signals was carried out using a Carl Zeiss LSM 700 laser scanning microscope equipped with intuitive ZEN 2009 software. Approximately, five to ten randomly selected fields were used for quantification using NIH ImageJ software. Cardiac interstitial fibrosis was measured and quantified by picrosirius red staining using five to six sections from each heart in chow and HFD groups in both wild-type and PRAK−/− mice. In addition, H&E staining was carried out according to an established laboratory protocol to estimate the myocyte size of each cross section. An approximate 300 myocytes per group obtained mid-distance from the base to apex were randomized to compare myocyte cross-sectional area in each group.
4
Inhibition of Pseudomonas aeruginosa Biofilm by Fe-Tart
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Example 4
Materials and Methods
Pseudomonas aeruginosa PAO-1 strain was routinely grown on either LB (Luria-Bertani, Oxoid, UK) agar plates at 37° C. or in broth at 37° C. with 200 rpm shaking. UV-sterilized glass slides were incubated in either 15 mL RPMI-1640 defined medium (Sigma, UK) or 15 mL RPMI-1640 with Fe-Tart inoculated with diluted (OD600=0.01) bacteria from overnight cultures at 37° C. with 60 rpm shaking for 72 hours. The slides were removed from bacterial culture and washed with 15 mL phosphate buffered saline at room temperature for 5 minutes three times and then rinsed with distilled H2O. After washing, the slides were stained with 20 μM SYTO17 dye (Invitrogen, UK) at room temperature for 30 minutes. After removing excess staining dye and air-drying, the samples were examined using a Carl Zeiss LSM 700 Laser Scanning Microscope with ZEN 2009 imaging software (Carl Zeiss, Germany). The coverage rate of bacteria on the surface was analysed using open source Image J 1.44 software (National Institute of Health, US).
Results
5
Immunofluorescence Staining of Drosophila S2 Cells
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Drosophila S2 cells were plated on ibidi dishes (Planegg/Martinsried, Germany) or coverslips were fixed with 3% paraformaldehyde solution (pH 7.3) at 21°C for 10 min. In some cases, fixed cells were subsequently permeabilised by treatment with 0.1% Triton X-100 at 21°C for 10 min. In cases where antigen retrieval was required, proteins were denatured by the treatment of fixed and permeabilised cells with 6 M guanidinium hydrochloride (GdnHCl) at 21°C for 7 min. Cells were subsequently rinsed with either PBS in the case of non-detergent-treated cells or in the case of detergent-treated samples, with 0.01% Triton X-100 in PBS (PBST). Washed cells were incubated with mouse monoclonal anti-PrP antibody 4H11 [55 (link)] and the Golgi-specific rabbit polyclonal antibody GM130 (Abcam, Cambridge, U.K.) at 37°C for 60 min. After three washing steps in PBS or PBST, as appropriate, cells were incubated with Alexa Fluor-488-conjugated goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany) or Cy3-conjugated goat anti-rabbit IgG (Dianova, Hamburg, Germany) at 21°C for 60 min. Nuclei were stained with Hoechst DNA staining dye (Sigma, Taufkirchen, Germany). Confocal laser scanning microscopy was performed on an LSM 700 laser scanning microscope (Zeiss, Göttingen, Germany).
6
Measuring NMDAR-mediated Ca2+ Dynamics
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HEK293T cells seeded in 35 mm glass-bottomed culture dishes (MatTek Corporation) were transiently transfected with GRIN1 and either GRIN2A WT or mutants (Q891X, R920K, W1271X). Thirty six hours post transfection cells were washed with Tyrode solution (5mM HEPES, 136 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1.9 mM CaCl2, 5.6 mM glucose, buffered to pH 7.4 with Tris base). Cells were loaded with 10 μM Fluo-3AM diluted in Tyrode supplemented with 0.1% BSA for 45 min in the dark, followed by 2 washes with Tyrode-BSA and 2 washes with Tyrode alone. Loaded cells were then left for 15 min to ensure complete hydrolysis of the acetoxymethyl ester (AM) groups. Images were taken every 5 sec over a period of 15 min and collected on a Zeiss LSM-700 laser scanning microscope with a 63x oil immersion lens in a single track mode using excitation 488 nm and emission BP 505–530 filter sets.
7
Cardiomyocyte Imaging of GLUT4 and VAMP2
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Cardiomyocytes were cultured in M-199 medium on glass bottom culture dishes pre-coated with laminin and allowed to attach for one hour. The medium was then exchanged for fresh M-199 and incubation continued in the presence or absence of ROCK inhibitors for another one hour at 37°C. The cells were washed with ice-cold PBS twice and fixed with 3.7% paraformaldehyde for 20 min. They were permeabilized and blocked with 0.1% NP40 plus 2% BSA in PBS for 45 min at room temperature, then treated with anti-GLUT4 and anti-VAMP2 antibodies (Cell Signaling Technology) at 1∶200 dilution in the same buffer at 4°C overnight. Following three 10 min washes with PBS containing 0.1% NP40 plus 2% BSA, the cells were incubated with a secondary antibody conjugated to Alexa Fluor® 488 or Alexa Fluor® 555 at 1∶1000 for one hour at room temperature. Following another three 10-min washes, the cells were mounted with Prolong Gold Antifade reagents and observed with a Zeiss LSM700 laser scanning microscope.
8
Apoptotic Morphological Changes in MCF-7 and 3T3-L1 Cells
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Apoptotic morphological changes were identified by the 4′,6-diamidino-2-phenyl-indole (DAPI) staining of MCF-7 cells and differentiating 3T3-L1 cells, which had been treated with genistein at 50 μM for 48 h two days subsequent to reaching confluence. Each cell line was seeded on poly-L-lysine-coated slides and fixed with 4% methanol-free formaldehyde for 30 min. Mounting medium containing DAPI was dispersed over the entire slide. The mounted slides were stored at 4°C in the dark. Each slide was observed under an LSM700 laser scanning microscope equipped with Zen 2011 software (Carl Zeiss Microscopy GmbH, Jena, Germany).
9
Intracellular Pressure Measurement Protocol
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Direct measurements of intracellular pressure were made using the 900A micropressure system (World Precision Instruments), as described previously (Petrie and Koo, 2014 ). Briefly, a 0.5-μm micropipette (World Precision Instruments) was filled with 1 M KCl solution and the resistance of the circuit was set to zero or null. The micropipette was positioned with an MPC-325 micromanipulator (Sutter Instrument) within an environmental chamber (10% CO2 and 37°C) on a Zeiss LSM700 laser scanning microscope using a 32×, 0.4 NA Ph1 objective. To make an intracellular pressure measurement, the microelectrode was inserted through the plasma membrane at a 45° angle, maintained in the cytoplasm for ≥5 s, and then removed. The pressure was measured perinuclearly, between the nucleus and the leading edge. The cytoplasmic hydraulic pressure was calculated as the mean pressure over this time interval.
10
Protein Localization Analysis Protocol
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PLA analysis was performed according to the manufacturer's instructions (OLink Biosciences, Uppsala, Sweden). Briefly, cells were incubated for 1 h at 37 °C on poly-lysine coated slides, fixed with 2% formaldehyde and blocked with 2% of normal donkey serum. Primary antibodies (used at the same dilutions than for immunocytochemistry) were incubated overnight at 4 °C, cells were then incubated with PLUS and MINUS secondary PLA probes against both rabbit and mouse IgG. Hybridization and ligation steps followed the incubation, and then amplification was performed. After mounting with Duolink mounting medium, images were acquired using a LSM 700 Laser Scanning Microscope in the larger focal plane of each cell (Carl Zeiss). Quantification was done using ImageJ.51
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