Dm5000b epifluorescence microscope

Manufactured by Leica

The Leica DM5000B is an epifluorescence microscope designed for advanced imaging applications. It features high-quality optics and a modular design to accommodate a variety of imaging techniques, including fluorescence microscopy. The DM5000B is capable of delivering high-resolution images, but a detailed description of its core function and capabilities is not available while maintaining an unbiased and purely factual approach.

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Lab products found in correlation

Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions) Gf c filter, by Cytiva (1 mentions) Whatman, by Cytiva (1 mentions) Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti chicken igy h l, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Dfc365 fx camera, by Leica (1 mentions) α sma, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Mowiol 4 88, by Polysciences (1 mentions) Fm4 64 dye, by Thermo Fisher Scientific (1 mentions) Sp5 spectral confocal microscope, by Leica (1 mentions) Prism 6.0f software, by GraphPad (1 mentions) Axioskop, by Leica (1 mentions) Ab150085, by Abcam (1 mentions) Prolong glass antifade mountant with nucblue stain, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions) Filipin, by Merck Group (1 mentions)

Spelling variants of this product

DM5000B (434 citations) DM5000B microscope (256 citations) Epifluorescence microscope (146 citations)

11 protocols using dm5000b epifluorescence microscope

Microscopic Visualization of Mitochondrial Morphology

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Cells transformed with the plasmid YX232-mtGFP were grown O.N. as previously
described, collected and immobilized in the slides by adding 0.5% (w/v) agar
prior to microscopy. When used, MitoTracker Red CMXRos (Molecular Probes) was
added to the culture medium at a final concentration of 0.4 µg/mL and incubated
for 20 min at 37° C. For the assays with the anion channel inhibitor
4´-diisothiocyano-2,2´-disulfonic acid stilbene (DIDS), overnight grown cells
were harvested and incubated in growth medium with 0.5 mM of DIDS for 5 min
40 (link). Samples were analysed on a Leica
Microsystems DM-5000B epifluorescence microscope with appropriate filter
settings using a 100 x oil-immersion objective. Images were acquired with a
Leica DCF350FX digital camera and processed with LAS AF Leica Microsystems
software (Leica Mycrosystems).

Trindade D., Pereira C., Chaves S.R., Manon S., Côrte-Real M, & Sousa M.J. (2016). VDAC regulates AAC-mediated apoptosis and cytochrome c release in yeast. Microbial Cell, 3(10), 500-510.

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Isolation and Analysis of Grape Plastids

Cited 1 time

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Plastids from grape
berry skins and leaves at E-L 34 and E-L 38
were isolated as previously described.^{5 (link)} with minor modifications. Chlorophyll quantification was performed
according to Lichtenthaler and Wellburn.³⁰ Values were normalized by the total protein amount determined spectrophotometrically
by the Bradford assay.^{31 (link)} Fluorescence microscopy
images were acquired with a Leica Microsystems DM-5000B epifluorescence
microscope.

Teixeira A., Noronha H., Frusciante S., Diretto G, & Gerós H. (2023). Biosynthesis of Chlorophyll and Other Isoprenoids in the Plastid of Red Grape Berry Skins. Journal of Agricultural and Food Chemistry, 71(4), 1873-1885.

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Quantifying Yeast Cell Wall Structure

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Yeast cells were grown under the conditions described above, harvested at early exponential phase, suspended in PBS, incubated with 0.02% (w/v) Calcofluor White (CFW, Fluka, St. Louis, MO) for 30 min at room temperature, and washed 3 times with PBS. Cells were observed using a Leica Microsystems DM-5000B epifluorescence microscope with appropriate filter settings using a 100x oil-immersion objective. Images were acquired with a Leica DCF350FX digital camera and processed with LAS AF Leica Microsystems software.

Rego A., Mendes F., Costa V., Chaves S.R, & Côrte-Real M. (2020). Pkh1p-Ypk1p and Pkh1p-Sch9p Pathways Are Activated by Acetic Acid to Induce a Mitochondrial-Dependent Regulated Cell Death. Oxidative Medicine and Cellular Longevity, 2020, 7095078.

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Calcium-Mediated Growth and Viability of Grapevine Cells

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Cells of V. vinifera L. cv. Gamay Fréaux var. Teinturier, gently provided by Prof. Serge Delrot (University of Bordeaux, France), were grown in liquid mineral medium supplemented with 58 mM sucrose, according to Decendit and Merillon (1996) , on a rotatory shaker at 100 r.p.m., under an 8 h dark/16 h light (200 μmol photons m -2 s -1 ) photoperiod, at 24 °C. Cells were subcultured weekly by transferring 15 mL aliquots into 30 mL of fresh medium. To study the effect of calcium on cell growth, size and viability, cells were subcultured in medium supplemented with increasing concentrations of CaCl 2 : 1 mM (basal levels), 5, 10, 50 and 100 mM. Three biological replicates were performed per CaCl 2 concentration. Cell growth was determined through dry-weight measurements: aliquots (1-3 mL) were filtered through pre-weighed GF/C filters (Whatman), washed with deionized water and weighed after 24 h at 80 °C. Ten days after subculture, cell viability was tested with the fluorescent dye fluorescein diacetate (FDA, Sigma), in a Leica Microsystems DM-5000B epifluorescence microscope with appropriate filter settings. Images were acquired with a Leica DCF350FX digital camera and processed with LAS AF Leica Microsystems software. Cell size was estimated by measuring the average cell diameter.

Martins V., Garcia A., Costa C., Sottomayor M, & Gerós H. (2018). Calcium- and hormone-driven regulation of secondary metabolism and cell wall enzymes in grape berry cells. Journal of plant physiology, 231.

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Influenza Virus Infection Kinetics

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MDCK or A549 cells were grown to 90–95% confluence on 13 mm glass coverslips and incubated with H2N3 or H3N8 (MOI 5) for 10 min at 4°C to allow virus binding, followed by 10, 20 or 30 min incubation at 37°C. Virus was incubated with phospholipid or IM prior to cell infection. Cells were in 1% PFA for 20 min at 4°C. Fixed cells were then blocked in 1% BSA (Fisher Scientific, UK) for 1 h at room temperature and incubated with specific chicken H3 antiserum for 1 h at room temperature. Cells were then incubated with Alexa Fluor 488 (goat anti-chicken IgY (H + L), Invitrogen). Then coverslips were mounted with Prolong Gold Anti-Fade Reagent with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) and imaged using a Leica DM 5000B epifluorescence microscope.

Al-dalawi L., Dunham S.P, & Rauch C. (2022). Lipid biophysics and/or soft matter-inspired approach for controlling enveloped virus infectivity. Journal of the Royal Society Interface, 19(189), 20210943.

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Histological Analysis of Kidney Structure

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Harvested kidneys were bisected and harvested for RNA and protein, and fixed in 4% PFA for antibody staining and histological staining with Sirius red or Masson's trichrome. Paraffin sections (4 µm) were stained with Sirius red or Masson trichrome and imaged with polarized light and bright‐field illumination. For immunofluorescence, 7‐μm frozen sections were washed with ice‐cold 100% methanol, boiled in 10 mM citric acid (pH 6) for 20 min, and incubated with primary antibodies against αSMA (Millipore), PDGFR‐β (gift from B. Stallcup), or with biotin‐LTL (Vector Laboratories). Reactivity was detected using fluorescently labeled secondary antibodies. Sections were counterstained with DAPI (Sigma‐Aldrich), mounted in Mowiol 4–88 (Poly Sciences), and digital images acquired using a Leica DM5000B epifluorescence microscope and Leica DFC365FX camera. To quantify percent area stained for Sirius red, PDGFR‐β, and α‐SMA, we analyzed images from each of at least 4 biological replicates by batch processing using a macro created in Image J. Images were converted to 8‐bit grayscale, the threshold adjusted, and percent area measured. To quantify intact proximal tubules with brush borders, LTL‐positive tubules were counted by an observer blinded to the experimental conditions using Image J.

Basta J., Robbins L., Stout L., Prinsen M.J., Griggs D.W, & Rauchman M. (2020). Pharmacologic inhibition of RGD‐binding integrins ameliorates fibrosis and improves function following kidney injury. Physiological Reports, 8(7), e14329.

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Fluorescent Membrane Staining of Bacterial Cells

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Cells were imaged as previously described^{57 (link)}. Briefly, overnight cultures were diluted 1:1,000 into LB supplemented with antibiotics and IPTG. Cultures were grown and induced for 7–8 h, at which point cells were diluted to an OD₆₀₀ of 0.5 in 1× PBS, then membrane stain FM4–64 dye (Thermo Fisher Scientific) was added to a final concentration of 20 μg ml⁻¹. Then, 1% agarose pads in de-ionized water were cut into squares of approximately 20 × 20 mm and placed on microscope slides, and 2 μl of diluted cultures was spotted onto a glass coverslip, then gently placed onto the agarose pad. FM4–64 signal was visualized using a Leica DM5000b epifluorescence microscope with a 100×-brightfield objective under RFP fluorescence channel. Images were captured using a Spot Pursuit CCD camera and an X-cite 120 Illumination system. Each slide was imaged with at least 20 fields of view for each biological replicate. Cell lengths were processed using the Fiji plugin MicrobeJ^{58 (link)}, and data were visualized and analysed using R by quantifying the length of the curvilinear (medial) axis of detected cells.

Hsueh B.Y., Severin G.B., Elg C.A., Waldron E.J., Kant A., Wessel A.J., Dover J.A., Rhoades C.R., Ridenhour B.J., Parent K.N., Neiditch M.B., Ravi J., Top E.M, & Waters C.M. (2022). Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria. Nature microbiology, 7(8), 1210-1220.

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Fluorescent Imaging of Neuronal Dendrites

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Worms were picked at the life stages indicated, mounted on slides with 2% agarose pads, and immobilized with 600 μM levamisole. Initial screening, time-course studies, and rescue experiments were conducted using a 40× or 63× objective on a Zeiss Axioskop or Leica DM5000B epifluorescence microscope. Dendrites, gene expression patterns, and subcellular localization were imaged with a Leica SP5 spectral confocal microscope at 63× with 0.5 μm per step and Leica LAS software. Secondary, tertiary, and terminal (quaternary and senary) dendrites were counted from the PVD cell body to the posterior end separately on the dorsal side, the ventral side, or both. Although numerous researchers contributed to the primary screen, positive hits were independently verified by confocal microscopy by a single researcher who did not participate in the primary screen. Statistical tests were performed and graphs created with Prism 6.0f software (GraphPad Software, Inc.).

Antonacci S., Forand D., Wolf M., Tyus C., Barney J., Kellogg L., Simon M.A., Kerr G., Wells K.L., Younes S., Mortimer N.T., Olesnicky E.C, & Killian D.J. (2015). Conserved RNA-Binding Proteins Required for Dendrite Morphogenesis in Caenorhabditis elegans Sensory Neurons. G3: Genes|Genomes|Genetics, 5(4), 639-653.

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Characterization of Migrated Neural Crest Stem Cells

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Identity of migrated HFSCs was assessed by immunostaining against nestin as a neural crest stem cell marker and SOX10 as a neural crest cells marker [6 (link)] using mouse anti-nestin (1:50; Abcam, #ab6142) and rabbit anti-SOX10 (1:100; proteintech, 10422-1-AP) primary antibodies. Briefly, cells at passage 1 were seeded in a 4-well chambered cell culture slide and fixed with 4% paraformaldehyde. Following several washing steps, cells were blocked with 10% normal goat serum, 1% FBS and 0.1% Triton X-100 prepared in phosphate-buffered saline (PBS). Then, the primary antibodies were applied overnight at 4 °C. After washing cells and re-blocked with 3% bovine serum albumin for 10 min, cells were exposed to goat anti-mouse IgG AlexaFluor488 (1:1000, ThermoFisher, #A-11001) or goat anti-rabbit IgG AlexaFluor488 (1:1000, Abcam, #ab150085) secondary antibodies at room temperature for two hours. To counterstain the nuclei, the ProLong™ Glass Antifade Mountant with NucBlue™ Stain (Invitrogen, # P36985) was used to cover the chambers. Finally, immunofluorescent images were obtained using a Leica DM5000B epifluorescence microscope.

Mousavi S.M., Akbarpour B., Karimi-Haghighi S., Pandamooz S., Belém-Filho I.J., Masís-Calvo M., Salimi H., Lashanizadegan R., Pouramini A., Owjfard M., Hooshmandi E., Bayat M., Zafarmand S.S., Dianatpour M., Salehi M.S, & Borhani-Haghighi A. (2022). Therapeutic potential of hair follicle-derived stem cell intranasal transplantation in a rat model of ischemic stroke. BMC Neuroscience, 23, 47.

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Filipin Staining of Unesterified Cholesterol

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filipin staining was performed on free-floating sections of PFA-fixed mouse brains. Sections were permeabilized with 1% BSA, 0.15% glycine and 0.02% saponin in PBS for 1.5 h. Unesterified cholesterol was stained using 0.005% filipin from a stock solution of 0.1% filipin (Sigma) in 10% DMSO. The sections were washed three times with permeabilization solution, followed by three washes with PBS and were finally mounted with Mowiol and Dabco, and analyzed with a Leica DM5000B epi-fluorescence microscope (excitation filter BP340-380; dichromatic mirror 400; suppression filter LP425).

Wolf H., Damme M., Stroobants S., D'Hooge R., Beck H.C., Hermans-Borgmeyer I., Lüllmann-Rauch R., Dierks T, & Lübke T. (2016). A mouse model for fucosidosis recapitulates storage pathology and neurological features of the milder form of the human disease. Disease Models & Mechanisms, 9(9), 1015-1028.

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