Dmi6000 confocal microscope

Manufactured by Leica

Sourced in Germany

The Leica DMI6000 is a confocal microscope that enables high-resolution imaging of samples. It features a motorized and encoded stage, allowing precise control and positioning of the specimen. The microscope is equipped with a selection of objectives to accommodate various sample types and magnification requirements.

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Lab products found in correlation

Las af software, by Leica (2 mentions) Application suite advanced fluorescence, by Leica (2 mentions) Lasaf 2, by Leica (2 mentions) Tcs sp8, by Leica (1 mentions) Anti gfp, by Rockland Immunochemicals (1 mentions) Vectashield, by Vector Laboratories (1 mentions) J2 mab, by Techcomp Instruments (1 mentions) Pmg8043, by Thermo Fisher Scientific (1 mentions) Mnoggin, by Thermo Fisher Scientific (1 mentions) Mrspondin 1, by R&D Systems (1 mentions) Rock inhibitor, by Merck Group (1 mentions) Matrigel, by BD (1 mentions) Draq5, by BioLegend (1 mentions) Rabbit anti ha monoclonal antibody, by Cell Signaling Technology (1 mentions) Poly d lysine, by Merck Group (1 mentions) D8417, by Merck Group (1 mentions) P1110, by Solarbio (1 mentions) Triton x 100, by Merck Group (1 mentions) Dm5500b wide field microscope, by Leica (1 mentions) Smi 32p, by Fortrea (1 mentions)

Close spelling variants of this product

DMI6000 CS confocal laser scanning microscope Leica SP5-II inverted (DMI6000 based) confocal microscope Leica TCS SP5 DMI6000 CS Confocal Microscope SP8 Lightning Confocal DMI6000 microscope DMI6000 laser confocal scanning microscope Leica TCS SP5 DMI6000 confocal microscope DMI6000 microscope DMI6000 Confocal microscope

12 protocols using dmi6000 confocal microscope

Quantifying M Cells and GBS Interactions

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Images from intestinal ligated loops (2–3 per animal) were randomly acquired using the Leica DMI6000 confocal microscope. Areas of interest including M cells were acquired. Ten images (three experiments in triplicate) of cultured Caco-2 cells in each condition were randomly acquired using the Leica DMI6000 confocal microscope or the wide field Leica 500A confocal microscope for quantitative imaging.
M cells and GBS were quantified using immunofluorescence with the ImageJ software (RRID:SCR_003070). For each image, we applied a protocol to measure the total surface of M cells, enterocytes and GBS, as well as the surface of GBS associated with M cells or enterocytes. Collected measurement values were exported to Microsoft Excel. The relative frequency of GBS associated with M cells was determined as a percentage of the total GBS surface from each individual image.

Hays C., Touak G., Bouaboud A., Fouet A., Guignot J., Poyart C, & Tazi A. (2019). Perinatal hormones favor CC17 group B Streptococcus intestinal translocation through M cells and hypervirulence in neonates. eLife, 8, e48772.

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Imaging mRNA and Protein Dynamics

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For YOX1 mRNA imaging, cells were grown overnight in synthetic medium lacking methionine in order to induce expression of the MS2 coat protein and then diluted in low-fluorescence medium containing methionine 4–6 h prior to imaging. Where indicated, cells were treated with HU for 2 h. Cells were imaged on a Leica DMI6000 confocal microscope.
For Yox1-GFP protein imaging, cells were first grown in synthetic complete medium overnight in a 96-well plate and diluted the next day for a second overnight culture in low-fluorescence synthetic complete medium. Cells were finally diluted to an OD₆₀₀ of 0.05 in fresh low-fluorescence synthetic complete medium in a 384-well imaging plate 2 h prior to imaging and incubated at 30 °C. Cells were imaged on an Opera confocal fluorescence microscope (PerkinElmer) in absence of drug and 2 and 4 h after addition of HU at a final concentration of 200 mM.
For Rnr3-GFP protein imaging, cells in exponential phase grown in complete low-fluorescence medium for 17–19 h were imaged on a Leica DMI6000 confocal microscope.

Loll-Krippleber R, & Brown G.W. (2017). P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance. Nature Communications, 8, 558.

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3D Reconstruction of Dental Stem Cell Niche

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For dentin culture assay, immunofluorescence micrographes were captured using A Leica DMI6000 confocal microscope with a Leica TCS SP8 attachment at a scanning thickness of 1 μm per section. Microscope was running LAS AF software from Leica. These images were used to reconstruct the growth of the cells into the dentine tubules using Imaris (version 9.0.2) (Bitplane). The three-dimensional (3D) reconstruction of the CL and surrounding transit amplifying mesenchyme region was performed by taking serial immunofluorescent-labeled cryosections. The epithelium was outlined from approximately 20 consecutive 20-µm-thick sagittal sections of the apical end of the mouse incisor.
The corresponding TAC region was defined by drawing a straight line between the epithelial tip, where marks boundary the epithelial stem cells and TACs on serial sections containing CL. 3D reconstruction of the region was performed with computational reconstruction using BioVis software (version: 3.1.1.11) (http://www.biovis3d.com). The Ki67-positive cells were then quantified on each section and total cell number were counted based on DAPI staining.

Walker J.V., Zhuang H., Singer D., Illsley C.S., Kok W.L., Sivaraj K.K., Gao Y., Bolton C., Liu Y., Zhao M., Grayson P.R., Wang S., Karbanová J., Lee T., Ardu S., Lai Q., Liu J., Kassem M., Chen S., Yang K., Bai Y., Tredwin C., Zambon A.C., Corbeil D., Adams R., Abdallah B.M, & Hu B. (2019). Transit amplifying cells coordinate mouse incisor mesenchymal stem cell activation. Nature Communications, 10, 3596.

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Quantifying DNA Repair Responses

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Following cisplatin damage, cells were either processed immediately, or medium was replaced and DNA repair allowed for indicated periods. Cell extraction was carried out in situ by washes of 0.1% Nonidet P-40 for 10 minutes on ice to remove all soluble proteins. Following fixation in 4% paraformaldehyde and cell permeabilization with 0.3% Triton X-100, cells were blocked overnight in 10% donkey serum at 4 °C. After incubation with indicated primary and secondary antibodies, cells were mounted with Prolong Gold antifade. Proximity ligation assay (DuoLink, Sigma) was performed using the manufacturer’s instructions. All fluorescence images were obtained using a Leica DMI 6000 confocal microscope using ×100 objective (1.4 numerical aperture) with LAS AF 2.7.2.9586 software (Leica Application Suite Advanced Fluorescence). Maximum intensity images from focal plane z-stacks (spaced 0.2 µm apart) were acquired and deconvoluted. Quantification of fluorescent signal were performed using Image J software.

Jarrett S.G., Carter K.M., Shelton B.J, & D’Orazio J.A. (2017). The melanocortin signaling cAMP axis accelerates repair and reduces mutagenesis of platinum-induced DNA damage. Scientific Reports, 7, 11708.

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Immunostaining and Confocal Imaging of Fly Brains

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We dissected fly brains in 19°C S2 medium, and fixed them in 1% paraformaldehyde at 4°C overnight. Fixed brains were washed 3 times for 30–60 min with PAT3 (0.5% Triton X-100 and 0.5% Bovine Serum Albumin (BSA) in PBS), then blocked with 3% NGS in PAT3 for 1.5 hours at room temperature. We incubated brains with primary and secondary antibodies as previously described 59], and mounted them in VectaShield (Vector Labs). We stained with anti-GFP (Rockland) at a dilution of 1:1000 along with nc82 antibody (DSHB) at 1:50 to label neuropil. We imaged brains using a 20× 1.0 NA objective on an Inverted Leica DMI 6000 confocal microscope with 1 or 1.5 μm separating each optical slice.

Ferris B., Green J, & Maimon G. (2018). Abolishment of spontaneous flight turns in visually responsive Drosophila. Current biology : CB, 28(2), 170-180.e5.

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Quantifying Virus-Induced Cytoplasmic Alterations

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BS-C-1 cells were mock-infected or infected with 5 PFU/cell of virus for 8 and 12 h, after which the cells were fixed and stained essentially as described above. The J2 MAb (SCICONS, Szirak, Hungary) was diluted 1:100 and incubated with the cells overnight. After washing, secondary anti-mouse Alexa Fluor 488 was applied at a 1:100 dilution for 6 h. Nuclei were stained with DAPI as above. Confocal images were collected using a Leica DMI6000 confocal microscope enabled with 40× oil immersion objective NA 1.32. Images were acquired using constant laser intensity for Argon Laser and 488 nm wave length for Alexa 488 excitation and photons were collected using constant photomultiplier electronic gain between the samples to quantify the differences in absolute intensity levels. Images were collected and processed using Imaris 7.1 (Bitplane AG, Zurich, Switzerland) and Adobe Photoshop CS3 (Adobe Systems, San Jose, CA) to adjust brightness. For quantification, images were collected using an automated tiling method to obtain an unbiased data pool from 50 to 100 tiles. Acquired images were further analyzed using Imaris image processing software to calculate the absolute total intensity per cell. Cumulative intensities were plotted as average mean intensity normalized to the uninfected sample.

Liu S.W., Katsafanas G.C., Liu R., Wyatt L.S, & Moss B. (2015). Poxvirus Decapping Enzymes Enhance Virulence by Preventing the Accumulation of dsRNA and the Induction of Innate Antiviral Responses. Cell host & microbe, 17(3), 320-331.

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Isolation and Culturing of Intestinal Crypts

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Crypt isolation was performed as described previously [23 (link)]. Briefly, Intestines were harvested and washed with 1× PBS. Villi were scraped and the remaining tissue were cut into 2 cm pieces and incubated in 2 mM EDTA/PBS for 30 min at 4 °C. Crypts were then collected by shaking and were cultured in Matrigel (BD Bioscience; Heidelberg, Germany; #356231) overlaid with medium containing 50 ng/mL EGF (Life technologies; Darmstadt, Germany; PMG8043), 100 ng/mL mNoggin (Peprotech, #250-38, Cranbury, NJ, USA), 1 μg/mL mR-spondin1 (R&D systems, #2474-RS-050, Minneapolis, MN, USA) (ENR) in the presence of 10 μM Rock-inhibitor (Sigma, Y0503, Setagaya City, Tokyo). Crypts were plated in 8-well Ibidi treat^® dishes and imaging was performed on a Leica DMI 6000 confocal microscope equipped with an incubation system and image analysis was completed using Leica LAS AF software (Frankfurt, Germany).

Tarquis-Medina M., Scheibner K., González-García I., Bastidas-Ponce A., Sterr M., Jaki J., Schirge S., García-Cáceres C., Lickert H, & Bakhti M. (2021). Synaptotagmin-13 Is a Neuroendocrine Marker in Brain, Intestine and Pancreas. International Journal of Molecular Sciences, 22(22), 12526.

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Proximity Ligation Assay for Protein Interactions

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Following damage, cells were either processed immediately or medium was replaced and DNA repair allowed for indicated periods. Cell extraction was carried out in situ by washes of 0.1% Nonidet P-40 for 10 min on ice to remove all soluble proteins. Following fixation in 4% paraformaldehyde and cell permeabilization with 0.3% Triton X-100, cells were blocked overnight in 10% donkey serum at 4°C. After incubation with indicated primary and secondary antibodies, cells were mounted with Prolong Gold antifade. Proximity ligation assay (DuoLink, Sigma) was performed using the manufacturer's instructions and as previously described (5 (link)). All fluorescence images were obtained using a Leica DMI 6000 confocal microscope using ×100 objective (1.4 numerical aperture) with LAS AF 2.7.2.9586 software (Leica Application Suite Advanced Fluorescence). Maximum intensity images from focal plane z-stacks (spaced 0.2 μm apart) were acquired and deconvoluted. Quantification of fluorescent signals was performed using Image J software.

Jarrett S.G., Wolf Horrell E.M, & D'Orazio J.A. (2016). AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair. Nucleic Acids Research, 44(22), 10711-10726.

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Immunofluorescent Localization of HA-tagged OPRM1

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OPRM1‐expressing or WT HT1080 cells were seeded at 7.5 × 10⁵ onto cover glass precoated with 0.1 mg mL⁻¹ Poly‐D‐Lysine (Sigma P7280). Cells were first washed with Hank's Balanced Salt Solution (Fisher Scientific MT21022CV), and then fixed with 4% paraformaldehyde/PBS for 15 minutes followed by quenching with 0.1 mol L⁻¹ glycine for 15 minutes. Afterward, the cells are washed 3x with 1X PBS and then blocked in 1%BSA/TBST for 1 hour. Rabbit monoclonal anti‐HA antibody (Cell Signaling 3724S) is used to stain the cells overnight, followed by goat anti‐rabbit Alexa Fluor 488 (Thermo Fisher A11070) and counterstained with Draq5 (Biolegend 424101) at 1:200 dilution. Slides were mounted and visualized using Leica DMI6000 confocal microscope using 63× oil‐immersion objective, with image processing using ImageJ software.

Hsu T., Mallareddy J.R., Yoshida K., Bustamante V., Lee T., Krstenansky J.L, & Zambon A.C. (2019). Synthesis and pharmacological characterization of ethylenediamine synthetic opioids in human μ‐opiate receptor 1 (OPRM1) expressing cells. Pharmacology Research & Perspectives, 7(5), e00511.

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Immunoconfocal Analysis of Fibroblasts

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Immunoconfocal analysis was used to identify the isolated fibroblasts. Cells were plated in glass-bottom culture dishes at a density of 1.5 × 10⁵ per well. After 24 h of incubation, cells were fixed with 4% paraformaldehyde (Solarbio, P1110) for 15 min at room temperature and then washed 3 times with PBS, and 0.3% Triton X-100 (Sigma, Triton™ X-100) was added for incubation for 15 min at room temperature. Afterwards, the cells were washed 3 times with PBS, incubated with 5% bovine serum albumin at room temperature for 2 h. Cells were incubated with primary antibody against Vimentin (CST, 5741S, 1:500) overnight at 4°C. On the next day, the cells were washed 3 times with PBS followed by incubation with goat anti-Rabbit fluorescein secondary antibody (ThermoFisher, A-11034, 1:1000) for 1 h at room temperature avoiding light exposure. DAPI was incubated with the cells at a concentration of 5 mg/L (Sigma, D8417) for nuclear staining away from light at room temperature for 5 min. Photographs were taken using a Leica DMI6000 confocal microscope with 63 × oil immersion lens and processed by LAS AF software.

Liu Z., Pei Y., Zeng H., Yang Y., Que M., Xiao Y., Wang J, & Weng X. (2021). Recombinant TSG-6 protein inhibits the growth of capsule fibroblasts in frozen shoulder via suppressing the TGF-β/Smad2 signal pathway. Journal of Orthopaedic Surgery and Research, 16, 564.

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