HUVECs were grown to confluence in 12 mm diameter coverslips, previously coated with 0.2% gelatin (Sigma-Aldrich) in PBS. Cells were fixed with 3% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 during 10 min at room temperature. HUVECs were blocked with PBS containing either 2% BSA (galectin-3) or 1% BSA plus 5% normal goat serum [NGS] (TRIM21). In order to monitor the co-localization of endoglin with TRIM21 or galectin-3, samples were incubated first with the mouse monoclonal antibody anti-human endoglin (P4A4; sc-20072, Santa Cruz Biotech). After washing twice with PBS, samples were incubated with antibodies, anti-TRIM21 (rabbit mAb #92043, Cell Signaling Technology) or anti-galectin-3 (rabbit polyclonal IgG, #PA5-34819, Thermo Fisher Scientific). Then, after several washing steps with PBS, cells were incubated with the secondary antibodies Alexa 488 goat anti-mouse IgG or Alexa 647 goat anti-rabbit IgG. To visualize the samples and for nuclear staining, slides were mounted in Prolong-DAPI (4′,6-diamidino-2-phenylindole) reagent (Molecular Probes, Invitrogen). Samples were observed using a SP5 fluorescence confocal microscope (DMI6000 CS Leica Microsystems).
Dmi6000 cs
Manufactured by Leica
Sourced in Germany
The DMI6000 CS is a high-performance inverted microscope designed for advanced cell culture and live-cell imaging applications. It features a compact and modular design, providing a stable and versatile platform for a wide range of imaging techniques, including transmitted light, fluorescence, and phase contrast microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Tsc sp5 confocal microscope, by Leica (3 mentions)
Las af acquisition software, by Leica (3 mentions)
Tcs sp5 2, by Leica (3 mentions)
Easy3d module, by Abberior (2 mentions)
Hxc apo, by Leica (2 mentions)
Onefive katana 06 hp, by NKT Photonics (2 mentions)
Pdl 800 d, by PicoQuant (2 mentions)
Tcs sp5, by Leica (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Dfc340fx, by Leica (2 mentions)
Fluorescence mounting medium, by Agilent Technologies (2 mentions)
Water immersion objective lens, by Leica (2 mentions)
Syto9, by Thermo Fisher Scientific (2 mentions)
Polymer coverslip, by Ibidi (2 mentions)
Dfc350fx, by Leica (2 mentions)
Tcs sp5 microscope, by Leica (2 mentions)
Las af software, by Leica (2 mentions)
Anti galectin 3, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Fd 10, by Merck Group (1 mentions)
35 protocols using dmi6000 cs
1
Endoglin Colocalization with TRIM21 and Galectin-3
2
Membrane Permeabilization Assay with GUVs
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GUVs were mixed with the
buffer (10 mM MES, 150 mM NaCl, pH 6.5, or 20 mM Tris, 150 mM NaCl,
pH 8.0), fluorescent dextran of 10 kDa in size (Sigma; FD10, final
concentration of 1 mg/mL) and proteins in the following combinations:
50 nM Y406A alone, 5 μM D22M alone or premixed 50 nM Y406A +
5 μM D22M. Mixtures were incubated for 30 min at room temperature
before imaging. Images were recorded on DMI6000 CS inverted microscope
with TCS SP5 laser scanning system (both Leica Microsystems, Germany)
with a 63 × oil-immersion objective (numerical aperture = 1.4).
The rhodamine-containing GUV membrane was excited at 550 nm, and emission
was detected from 570 to 600 nm. FD10 were excited at 488 nm, and
emission was detected from 497 to 527 nm. Percent of permeabilization
was calculated from green channel fluorescent intensities in ImageJ
software, namely fluorescent intensities inside the vesicles were
divided by background intensities outside the vesicles. For each condition
50 to 500 GUVs were analyzed.
buffer (10 mM MES, 150 mM NaCl, pH 6.5, or 20 mM Tris, 150 mM NaCl,
pH 8.0), fluorescent dextran of 10 kDa in size (Sigma; FD10, final
concentration of 1 mg/mL) and proteins in the following combinations:
50 nM Y406A alone, 5 μM D22M alone or premixed 50 nM Y406A +
5 μM D22M. Mixtures were incubated for 30 min at room temperature
before imaging. Images were recorded on DMI6000 CS inverted microscope
with TCS SP5 laser scanning system (both Leica Microsystems, Germany)
with a 63 × oil-immersion objective (numerical aperture = 1.4).
The rhodamine-containing GUV membrane was excited at 550 nm, and emission
was detected from 570 to 600 nm. FD10 were excited at 488 nm, and
emission was detected from 497 to 527 nm. Percent of permeabilization
was calculated from green channel fluorescent intensities in ImageJ
software, namely fluorescent intensities inside the vesicles were
divided by background intensities outside the vesicles. For each condition
50 to 500 GUVs were analyzed.
3
Immunofluorescence Detection of MAM-7 in L. rhamnosus
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To asses if recombinant L. rhamnosus strains expressed MAM-7, bacterial samples were placed onto coverslips previously treated with poly-l -lysine (Sigma) for 20 min at room temperature. Each coverslip with the sample was dried at 37 ℃ and fixed with 3 % paraformaldehyde (PFA, Merck) for 15 min at room temperature. Then, coverslips were put on 24-well cell culture plate (TrueLine) and washed three times with PBS 1 × (Gibco). Samples were then blocked using 1 % albumin bovine serum (BSA, Calbiochem®) for 1 h at room temperature in humidity chamber. Each sample was incubated with 50 μL of 1:50 anti-Vibrio primary antibody (BacTrace anti-vibrio species antibody, KPL) for 12 h at 4 ℃. Once time had passed, each sample was washed with PBS 1 × (Gibco) and then, incubated with 50 μL of 1:500 goat anti-rabbit secondary antibody alexa fluor® 488 conjugate (molecular probes) in darkness and humidity chamber for 2 h. After that, samples were washed three times with PBS (Gibco) and coversilps were mounted over microscope slides with Dako fluorescent mounting medium (Fluoromont-g Emsdiasum) for 1 h in darkness. Image adquisition was performed in Leica TCS SP8, DMI6000 CS (Leica Microsystems). Adquisition was performed with argon laser, HC PL APO CS2 63x/1.40 oil objective, and optic slices were set at 0.895 μm.
4
Immunocytochemistry and Immunohistochemistry Protocol
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Cells in microfluidic devices were washed with PBS (100 mM, pH 7.4) and fixed in PFA (4% w/v) for 25 min, washed and permeabilized with triton X-100 (0.2% v/v in PBS) for 30 min with gentle shaking. Cells were then treated with a blocking solution (BSA, 3% w/v in PBS) for 30 min and incubated overnight at 4 ∘C with mouse anti-β-III Tubulin (1:200) antibody. DyLight 488 tagged horse anti-mouse IgG antibodies were used to detect primary antibody binding, followed by Hoechst nuclear counterstain, and visualized under an inverted confocal laser scanning microscope (DMI6000 CS, Leica Microsystems GmbH, Germany). Non-specific IgG primary antibody was used as controls. Further, immunohistochemical studies were performed on 4% PFA fixed skin sections to determine skin nerve fibre binding of IB4-AuNP. Briefly, 5 µm thin skin sections were permeabilized using Tris-buffer (pH 7.5), and blocked with BSA (1% w/v), for 2 h at RT. Sections were then co-treated with mouse anti-PGP9.5 antibody (1:500), and DyLight 594 tagged IB4-AuNP, overnight at 4 ∘C. Sections were then counterstained with DyLight 488 horse anti-mouse secondary antibody, mounted and visualized under an inverted fluorescence microscope (DMI3000 B, Leica Microsystems, Germany).
5
Super-Resolution Microscopy Setup
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We used a custom-built STED/confocal setup55 (link) constructed around an inverted microscope body (DMI 6000 CS, Leica Microsystems) which was equipped with a TIRF oil objective (x100, 1.47 NA, HXC APO, Leica Microsystems) and a heating box (Cube and Box, Life Imaging Services) to maintain a stable temperature of 32 °C. A pulsed-laser (PDL 800-D, PicoQuant) was used to deliver excitation pulses at 488 nm and a de-excitation laser (Onefive Katana 06 HP, NKT Photonics) operating at 594 nm was used to generate the STED light pulses. The STED beam was profiled to a donut shape using a spatial light modulator (Easy3D Module, Abberior Instruments). Image acquisition was controlled by the Inspector software (Abberior Instruments). The spatial resolution of the microscope was 175 nm (x-y) and 450 nm (z) in confocal mode and 60 nm (x-y) and 160 nm (z) in STED mode.
6
Differentiation of SH-SY5Y Cells into Neurons
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SH-SY5Y cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), non-essential amino acids (0.1 mM), sodium bicarbonate (3.6 g/L), sodium pyruvate (1 mM), L-glutamine (2 mM), and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) in a 5% CO2 humidified incubator at 37°C. After 24 h of cell plating, differentiation was induced by lowering the FBS in culture medium to 3% and adding 10 μM all-trans retinoic acid (RA) (Sigma, R2625) and 0.5% B-27 supplement (Thermo Fisher Scientific, 17504044) during 4 days, changing the medium every day. To evaluate the differences in cell morphology in proliferative and differentiated cells, we analyzed cells under phase-contrast light microscopy using a 20x oil immersion objective. Images were captured with epifluorescence microscopy (Leica Microsystems, DMI6000CS). To validate our neuronal cell model, we used three neuron-specific markers; NeuN, beta III tubulin and NSE. We observe cells that acquire the neuronal phenotype in the presence of retinoic acid and express neuronal markers.
7
3D Super-Resolution Microscopy with Home-Built STED Setup
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We used a home-built 3D-STED setup (for details, see Inavalli et al., 2019 (link)) constructed around an inverted microscope body (DMI 6000 CS, Leica Microsystems), which was equipped with a TIRF oil objective (100×, 1.47 NA, HXC APO, Leica Microsystems) and contained within a heating box (Cube and Box, Life Imaging Services) to maintain a stable temperature of 32°C. A pulsed-laser (PDL 800-D, PicoQuant) was used to deliver excitation pulses (90 ps at 80 MHz) at 485 nm and a synchronized de-excitation laser (Onefive Katana 06 HP, NKT Photonics) operating at 592 nm was used to generate the STED light pulses (500–700 ps). The STED beam was reflected on a spatial light modulator (SLM, Easy3D Module, Abberior Instruments) to generate a mixture of doughnut-shaped and bottle-shaped beams for 2D and 3D-STED, respectively. Image acquisition was controlled by the Imspector software (Abberior Instruments). The performance and spatial resolution of the microscope was checked and optimized by visualizing and overlapping the PSFs of the laser beams using 150-nm gold nano-spheres and 40-nm fluorescent beads and correcting the main optical aberrations with the SLM. The spatial resolution was 175 nm (lateral) and 450 nm (axial) in confocal mode and 60 nm (lateral) and 160 nm (axial) in STED mode.
8
Confocal Microscopy of Neuronal Receptor Puncta
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Images for the above experiments were acquired using a Leica DMI 6000 CS inverted laser-scanning confocal microscope (argon/krypton laser) with a Plan Apo 63x/glycerol immersion objective. The laser intensity, photomultiplier gain, and iris were optimized for each experiment and kept constant within that experiment. A Z-series of twenty optical sections (0.2 μm/section) was collected to capture three dimensional information and the series was reconstructed to yield images. For the insertion experiments, the images were analyzed using NIH ImageJ 1.43 software. For each image, the anti-β2/3 subunit fluorescence signal was analyzed for fluorescence intensity, number of puncta and puncta size (range 0.1–2.5 μm). Each experiment was performed in triplicate with at least two images acquired for each triplicate dish to yield six images for each experimental condition per experiment. All quantified data were acquired and analyzed by an investigator blinded to the experimental treatments.
9
Super-Resolution Microscopy of Nematodes
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Super‐resolution microscopy was performed using the Leica TCS SP8 gated STED. This instrument is an inverted microscope (Leica DMI6000 CS) equipped with a 100× oil objective lens and a White Light Laser (WLL). Images were taken with a resolution of about 60 nm (pixel size of 18.93 × 18.93 nm) following the use of a depletion laser at 592 nm. In all the experiments, nematodes were paralyzed with 25 mM Levamisole (AppliChem) in M9 buffer mounted on 2% agarose pads on glass slides and closed with coverslips. FRAP experiments were conducted blind to the respective condition (genotype, treatment, or RNAi) of the nematodes.
10
Immunofluorescence Staining Protocol
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One × 104 cells were seeded in 12-well cell culture plates and transfected with each siRNA as described. When needed, samples were also transfected after 24 h, using Lipofectamine LTX (Life Technologies). Next, cells were washed with PBS, fixed with ice-cold methanol for 10 min, permeabilized with 0.2% Triton X-100 solution for 20 min and then blocked for 20 min with 2.5% BSA in PBS. Cells were then incubated with appropriate primary antibodies for 30 min, washed three times with PBS, and then incubated with appropriate Alexa Fluor 488-conjugated (Invitrogen, A21202) or Alexa Fluor 555-conjugated (Invitrogen, A31570) secondary antibodies. Nuclei were stained with 1.5 μM 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 5 min. Coverslips were mounted in fluorescence mounting medium (Dako, S3023). Samples were visualized on a TSC SP5 confocal microscope (Leica, 5100000750) installed on an inverted LEICA DMI 6000CS (10741320) microscope using an oil immersion PlanApo 40 × 1.25 NA. Images were acquired using the LAS AF acquisition software (Leica).
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