Leica dm5000b microscope

Manufactured by Leica Microsystems

Sourced in Germany, United States, Israel, United Kingdom, Switzerland

The Leica DM5000B is a high-performance upright microscope designed for a wide range of scientific applications. It features an advanced optical system, providing users with high-resolution imaging and excellent contrast. The microscope is equipped with a range of illumination options and a versatile stage that accommodates a variety of sample types. The Leica DM5000B is a robust and reliable instrument that supports various imaging techniques, including brightfield, darkfield, and polarized light microscopy.

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61 protocols using leica dm5000b microscope

Cytoskeletal and Spindle Morphology Assays

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To check the actin cytoskeleton, rhodamine-phalloidin staining was performed according to the manufacturer’s instructions (Beyotime, China). Generally, cells were fixed with 4% formaldehyde (v/v) and then washed three times with PBS buffer containing 0.1% Triton. Afterwards, cells were incubated in rhodamine-phalloidin dye (1/40 diluted with PBS buffer containing 0.1% Triton) overnight at 4℃. Finally, cells were washed twice with PBS buffer and imaged on a Leica DM5000B microscope (Leica Microsystems) with a 100 × objective.
Spindle morphology assays were performed as previously described [32] (link). Generally, overnight cells were collected and fixed with 70% ethanol for 5 min, washed twice with 1 × PBS, incubated in 1.0 μg/ml DAPI for 10 min, and then washed twice with 1 × PBS before being mounted for analysis. Cells were imaged on a Leica DM5000B microscope (Leica Microsystems) with a 40 × objective.

Yao S., Feng Y., Islam A., Shrivastava M., Gu H., Lu Y., Sheng J., Whiteway M, & Feng J. (2020). Loss of Arp1, a putative actin-related protein, triggers filamentous and invasive growth and impairs pathogenicity in Candida albicans. Computational and Structural Biotechnology Journal, 18, 4002-4015.

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Quantifying Cardiac Oxidative Stress

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Fresh mouse heart samples were washed with PBS and then quickly frozen in liquid nitrogen. The frozen heart tissues were embedded into OCT, and then cut into 5 μm frozen sections. After soaked in PBS for 1 h at room temperature, the sections were incubated with 5 μmol/L DHE for 30 min at 37 °C in dark, followed by washing with PBS and photographed by Leica DM5000B microscope (Leica Microsystems, Wetzlar, Germany) or used for ROS detection by the Reactive Oxygen Species Fluorometric Assay Kit (Elabscience, Wuhan, China). The fluorescence intensity was quantified using the Image J software.

Yang Y., Feng K., Yuan L., Liu Y., Zhang M., Guo K., Yin Z., Wang W., Zhou S., Sun H., Yan K., Yan X., Wang X., Duan Y., Hu Y, & Han J. (2022). Compound Danshen Dripping Pill inhibits hypercholesterolemia/atherosclerosis-induced heart failure in ApoE and LDLR dual deficient mice via multiple mechanisms. Acta Pharmaceutica Sinica. B, 13(3), 1036-1052.

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Immunofluorescence Microscopy Protocol

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Indirect immunofluorescence microscopy was performed as described previously (70 (link)), using rabbit anti-FLAG polyclonal antibody (1/1000 dilution) (66 (link)) and mouse anti-HA mAb (HA-7; 1/400 dilution; Merck) as primary antibodies and Alexa Fluor 488-conjugated anti-rabbit IgG antibody and Alexa Fluor 594-conjugated anti-mouse IgG antibody (1/200 dilution each; Thermo Fisher Scientific) as secondary antibodies. Cells were mounted on glass microscope slides with ProLong Gold Antifade reagent (Thermo Fisher Scientific) and observed under a Leica DM5000B microscope (Leica Microsystems).

Kato R., Takenaka Y., Ohno Y, & Kihara A. (2024). Catalytic mechanism of trans-2-enoyl-CoA reductases in the fatty acid elongation cycle and its cooperative action with fatty acid elongases. The Journal of Biological Chemistry, 300(2), 105656.

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Immunoblotting and Immunofluorescence of Cell Signaling

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For immunoblotting, cells were lysed in RIPA buffer, followed by centrifugation, and subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were immunoblotted with various primary antibodies [DAL-1 (Abnove, Taiwan), α-1-catenin (Abcam, Cambridge, MA, USA), β-catenin (Abcam), N-cadherin (BD Biosciences, Bedford, MA, USA), vimentin (BD Biosciences) and β-actin (Zhongshan Biotech Co., Beijing, China)], followed by the corresponding fluorescent-conjugated anti-mouse or anti-rabbit antibodies (Rockland Immunochemicals Inc., Gilbertsville, PA, USA). The fluorescence signals were visualized using an Odyssey Infrared Imaging System (Li-COR, USA). For immunofluorescence, cells were seeded onto coverslips in six-well plates, and then were fixed in 4% paraformaldehyde. The coverslips were coated with primary antibodies against DAL-1 and β-catenin, followed by incubation with the anti-mouse or anti-rabbit antibodies respectively, and 4′,6-diamidino-2-phenylindole (DAPI) staining. Images were obtained using a Leica DM5000B microscope (Leica Microsystems, Solms, Germany).

Wang H., Xu M., Cui X., Liu Y., Zhang Y., Sui Y., Wang D., Peng L., Wang D, & Yu J. (2016). Aberrant expression of the candidate tumor suppressor gene DAL-1 due to hypermethylation in gastric cancer. Scientific Reports, 6, 21755.

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Histological Analysis of Tibialis Anterior Muscles

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Tibialis anterior muscles were equilibrated at −20°C for 1 h prior to sectioning. A microtome cryostat was used to cut 10‐μm‐thick serial transverse sections, which were transferred to positively charged glass slides. The slides were then sequentially submerged in 100% ethanol for 1 min, 70% ethanol for 1 min, dH₂0 for 2 min, and Gill's Haematoxylin for 2 min. Sections were then washed thoroughly in dH₂0 followed by sequential submersions in the following solutions: Scott's Solution for 15 s, dH₂0 for 2 s, 70% ethanol for 1 min, Eosin for 2 min, 95% ethanol with gentle shaking for 1 min, 100% ethanol for 30 s, and Xylene for 3 min. Slides were allowed to dry for 30 min and then mounted with glass cover‐slips using Permount. All sections were visualized and images captured using a Leica DM5000B microscope (Leica Microsystems Bannockburn, IL) and the Leica Application Suite, version 3.5.0 software. This software was also used to trace and measure muscle fibre cross‐sectional area (CSA).

Kandarian S.C., Nosacka R.L., Delitto A.E., Judge A.R., Judge S.M., Ganey J.D., Moreira J.D, & Jackman R.W. (2018). Tumour‐derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumour‐bearing mice. Journal of Cachexia, Sarcopenia and Muscle, 9(6), 1109-1120.

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Microstructural Analysis of Oleogels

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The microstructure of the samples was studied using a Leica DM5000 B microscope (Leica Microsystems, Germany) with a color camera (Leica MC330 CCD). The oleogel samples were prepared on the center of a glass slide and then gently covered by a coverslip. After a night of cold storage, the samples were observed under 20× magnification and a polarizing microscope.

Wang L., Wen Y., Su C., Gao Y., Li Q., Du S, & Yu X. (2022). Effect of water content on the physical properties and structure of walnut oleogels. RSC Advances, 12(15), 8987-8995.

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Microscopic Imaging of Biological Samples

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After mounting and covering with glass cover slips, the object slides were optionally sealed with nail polish to protect the samples from drying and to prolong their durability, or they were immediately inspected under the microscope without sealing.
Images were captured with a Leica DM 5000 B microscope equipped with a DFC420 C camera and LAS-X-software (Leica Microsystems, Wetzlar, Germany) or with a confocal laser scanning microscope, either a SP5 (Leica Microsystems, Wetzlar, Germany) or a LSM 880 (Zeiss, Jena, Germany).

Kakoschke T.K., Kleinemeier C., Knösel T., Kakoschke S.C, & Ebel F. (2023). The Novel Monoclonal IgG1-Antibody AB90-E8 as a Diagnostic Tool to Rapidly Distinguish Aspergillus fumigatus from Other Human Pathogenic Aspergillus Species. Journal of Fungi, 9(6), 622.

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Histological Analysis of Skeletal Muscle

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Serial skeletal muscle sections (10 µM) were cut and transferred to positively charged glass slides. Morphology was evaluated in muscle sections from all patients using hematoxylin and eosin (H&E), Masson’s Trichrome (stains collagen) and Oil Red O (stains lipid). A subset of cachectic PDAC patients and non-cancer control subjects were further processed using Alizarin Red S (stains calcium), CD68 antibody (to label macrophages) and antibodies against UBIQUITIN, P62, LAMP1 and LC3 to assess pathways involved in cellular quality control. All images were obtained using a Leica DM5000B microscope (Leica Microsystems; Bannockburn, IL) and analyzed using ImageJ software. Detailed methods have been described by us previously (18 (link)) and can be found in Supplementary Methods (available online).

Judge S.M., Nosacka R.L., Delitto D., Gerber M.H., Cameron M.E., Trevino J.G, & Judge A.R. (2019). Skeletal Muscle Fibrosis in Pancreatic Cancer Patients with Respect to Survival. JNCI Cancer Spectrum, 2(3), pky043.

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Histological Analysis of Thymus and Spleen

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Thymi and spleens were fixed in neutral buffered formalin (NBF) overnight. Formalin-fixed tissues were paraffin embedded and sectioned, then stained with hematoxylin/eosin (H&E) and Picrosirius red. For H&E sections, digital images were acquired using an Aperio Scan Scope CS system (Aperio, Inc., Vista, California, United States). For Picrosirius red sections, digital images were acquired using a Leica DM 5000B microscope (Leica Microsystems, Inc., Buffalo Grove, Illinois, United States) with a Diagnostic Instruments Spot RTKE camera (SPOT Imaging Solutions, Sterling Heights, Michigan, United States) (Unthank et al. 2015 (link), Vogel et al. 2015 (link)). For quantifying the Picrosirius red stained thymus tissue, 4–6 images were obtained randomly per slide at 5X magnification. All images were analyzed using ImageJ (version 1.50v; National Institutes of Health). Red, green, and blue (RGB) images were converted to grayscale then percentage of red-stained collagen in the background of pale-stained medullar area was measured. More measurement information can be found on the ImageJ NIH website.

Wu T., Plett P.A., Chua H.L., Jacobsen M., Sandusky G.E., MacVittie T.J, & Orschell C.M. (2020). IMMUNE RECONSTITUTION AND THYMIC INVOLUTION IN THE ACUTE AND DELAYED HEMATOPOIETIC RADIATION SYNDROMES. Health physics, 119(5), 647-658.

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Stereological Counts of Cortical Neurons

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Unbiased stereological counts of neurons labeled using NeuN DAB immunohistochemistry was undertaken as previously described [36 (link)]. Briefly, contours of layers I, II/III, IV, V, VI were drawn at 5 × objective in the somatosensory and motor cortex, based on cell density and soma size. Optical fractionator sampling was performed on a Leica DM5000B microscope (Leica Microsystems, Bannockburn, IL) with a motorized stage at 40 × objective. Section thickness was assessed at each sampling site with a 2.5 µm guard zone at the top and bottom of the section. Grid size was (X) 220 µm, (Y) 166 µm and the counting frame was (X) 50 µm, (y) 50 µm, with a depth of 25 µm. Gunderson coefficients of error for m = 1 was less than 0.10 for each section. The Stereo Investigator software performed stereological estimates with equal parameters for each layer in Cx3Cr1-Cre, Cx3cr1-iDTR, and iDTR mice (n = 3 for each genotype).

Bedolla A., Taranov A., Luo F., Wang J., Turcato F., Fugate E.M., Greig N.H., Lindquist D.M., Crone S.A., Goto J, & Luo Y. (2022). Diphtheria toxin induced but not CSF1R inhibitor mediated microglia ablation model leads to the loss of CSF/ventricular spaces in vivo that is independent of cytokine upregulation. Journal of Neuroinflammation, 19, 3.

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