Dm5500b

Manufactured by Leica Microsystems

Sourced in Germany, United States

The DM5500B is a high-performance microscope system designed for advanced research and analysis applications. It features a motorized nosepiece, stage, and focus mechanism, allowing for precise control and automated workflows. The DM5500B provides exceptional optical quality and illumination for detailed imaging and analysis of a wide range of specimens.

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Lab products found in correlation

Vectashield plus antifade mounting medium with dapi, by Vector Laboratories (2 mentions) Lsm 700, by Zeiss (2 mentions) Permount, by Thermo Fisher Scientific (1 mentions) Ab55138, by Abcam (1 mentions) Vectashield hardset, by Vector Laboratories (1 mentions) Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions) Dfc365fx, by Leica Microsystems (1 mentions) Col 3, by Abcam (1 mentions) Glial fibrillary acidic protein (gfap), by Abcam (1 mentions) C11440, by Hamamatsu Photonics (1 mentions) Nile red, by Merck Group (1 mentions) Paraformaldehyde (pfa), by Shanghai Lingfeng Chemical Reagent (1 mentions) Dfc360 fx, by Leica camera (1 mentions) Depex, by Avantor (1 mentions) Las v4, by Leica Microsystems (1 mentions) Dfc550, by Leica Microsystems (1 mentions) Pathvision, by Abbott (1 mentions) Cytovision software, by Leica Microsystems (1 mentions) Rhodamine 123, by Merck Group (1 mentions) Sw55 rotor, by Beckman Coulter (1 mentions)

17 protocols using dm5500b

Motoneuron Counting in Spinal Cord

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For the motoneuron counting, cross-sections of the lumbar spinal cord were stained with Toluidine Blue (0.05% in distilled water), dehydrated diaphanized, and mounted with Permount (Fisher Scientific) and coverslip. The slides were analyzed under the microscope (DM5500B, Leica Microsystems), and the motoneurons located in the dorsolateral nuclei at lamina IX of Rexed of the ipsi- and contralateral sides of the spinal cord were counted. Approximately 20 alternate slices spaced 240 μm apart were used, and the counts were corrected using the Abercrombie formula [54 ]:

N = n t / (t + d)

where N is the corrected number of counted neurons, n is the number of counted neurons, t is the distance between the counted sections, and d is the mean neuron diameter. As the difference in cell size significantly affects the number of cells, the d-value was calculated specifically for each experimental group (ipsilateral and contralateral). In this sense, the diameter of 15 motoneurons for each group was measured using the Image J software (version 1.33u) and the mean was calculated.
Subsequently, the ipsi/contralateral ratio was calculated, which corresponds to the neuronal survival rate, and the mean ± standard error was calculated for each experimental group.

da Silva N.S., Lombardi J., Kirchhoff F., Ferreira RS J.r., Barraviera B., de Oliveira A.L, & Cartarozzi L.P. (2024). Effects of local and systemic treatment with human natural killer-1 mimetic peptide (HNK-1) after ventral root avulsion and reimplantation in mice. The Journal of Venomous Animals and Toxins Including Tropical Diseases, 30, e20230065.

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Histological Scoring of Renal Markers in Preeclampsia

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The human kidney sections were scored histologically by an experienced renal pathologist who was blinded with respect to the subjects’ clinical data. Each immunostained sample was evaluated and scored by two independent observers. Because the renal pathological manifestations of preeclampsia are present in the glomerulus, we scored the staining of the various markers in the glomerulus only, scoring ≥50 glomeruli per section. The immunostained complement components were scored semi-quantitatively as follows: 0 represents an absence of—or traces of—glomerular staining; 1 represents segmental glomerular staining; and 2 represents global staining of the glomeruli. If positive (score ≥1), the kidney sections were further classified as having either focal (10–50% of the glomeruli) or diffuse (>50% of the glomeruli) deposits. Caspase-3 staining was analyzed by counting the number of caspase-3–positive cells in 50 glomeruli and comparing the number of positive cells between the study groups. TUNEL staining was scored as absent or present. For immunofluorescence, the slides were analyzed for either the absence or presence of immune deposits in the glomeruli using both a fluorescence microscope (DM5500B, Leica Instruments) and a confocal laser-scanning microscope (LSM 700, Zeiss).

Penning M., Chua J.S., van Kooten C., Zandbergen M., Buurma A., Schutte J., Bruijn J.A., Khankin E.V., Bloemenkamp K., Karumanchi S.A, & Baelde H. (2015). CLASSICAL COMPLEMENT PATHWAY ACTIVATION IN THE KIDNEYS OF WOMEN WITH PREECLAMPSIA. Hypertension, 66(1), 117-125.

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Oral Cancer Tissue Analysis

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Frozen tissues of 15 patients with oral cancer were analysed to assess sensitivity and specificity. Normal tissue of the oral cavity of each patient was used as a negative control. Tissues were obtained from the Erasmus Medical Center (Rotterdam, The Netherlands) tissue bank. Cryosections were cut into slices of 10 μm. The cryosections were imaged by fluorescence microscopy (Leica microsystems DM5500 B, Eindhoven, The Netherlands) at 0, 1, 5 and 10 min after application of gGlu-HMRG (50 μl of 50 μM in PBS). Fluorescence intensity (negative, weak, or positive) was scored independently by two authors (MS & HH). Subsequently, each slice was stained using an anti-GGT1 antibody (dilution 1:800; ab55138, Abcam, Cambridge, UK). The staining of epithelium was also scored (negative, weak, or positive) by two authors (LH & HH). Concordance between fluorescence and GGT status was subsequently evaluated.

Slooter M.D., Handgraaf H.J., Boonstra M.C., van der Velden L.A., Bhairosingh S.S., Que I., de Haan L.M., Keereweer S., van Driel P.B., Chan A., Kobayashi H., Vahrmeijer A.L, & Löwik C.W. (2018). Detecting tumour-positive resection margins after oral cancer surgery by spraying a fluorescent tracer activated by gamma-glutamyltranspeptidase. Oral oncology, 78, 1-7.

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Immunofluorescence Staining of Rhodopsin in Retina

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These assays were performed as previously described.^{13 (link)} Sagittal sections (4-μm) of paraffin-embedded eyecups, cut through the optic nerve head, were either stained with hematoxylin and eosin (H&E) or prepared for immunofluorescence staining. Slides containing sections were incubated overnight with primary antibody at 4°C (1:400 anti-Rhodopsin monoclonal RetP1; Sigma-Aldrich Corp.), washed, and incubated with secondary antibody (1:500 AlexaFluor 568 goat-anti-mouse; Invitrogen Corp., Carlsbad, CA, USA) for 2 hours at room temperature. Coverslips were mounted with mounting medium with DAPI (Vectashield Hardset; Vector Laboratories, Inc., Burlingame, CA, USA). All imaging was performed using a microscope and camera (DM5500B and DFC365FX; Leica Microsystems GmbH, Buffalo Grove, IL, USA).

Ruzycki P.A., Linne C.D., Hennig A.K, & Chen S. (2017). Crx-L253X Mutation Produces Dominant Photoreceptor Defects in TVRM65 Mice. Investigative Ophthalmology & Visual Science, 58(11), 4644-4653.

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Mosquito Embryo Fertilization Assay

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Eggs were collected from a pool of 50 gravid females from each mating group over a period of 1 h in the first and second gonotrophic cycles and allowed to embryonate for additional 2 h. Embryo dechorionation, fixation and endochorion disruption was done as described.⁷⁴ (link) Following endochorion removal, 100 embryos from each mating group were transferred to a slide and stained with Vectashield Plus Antifade Mounting Medium with DAPI (Vector Laboratories) for 1 h. Slides were mounted and viewed at 100x magnification under a fluorescence microscope (DM5500B, Leica Microsystems). Eggs with a maximum of 4 nuclei were scored as unfertilized while those with 5 or more nuclei were scored as fertilized in our assay. Scoring was conducted by an observer who was blinded to the genotype of the embryos. This experiment was conducted across three batches of mosquitoes. For each batch, all genotypes were reared in parallel with the same feeding and environmental conditions.

David O.G., Sanchez K.M., Arce A.V., Costa-da-Silva A.L., Bellantuono A.J, & DeGennaro M. (2023). Fertility decline in female mosquitoes is regulated by the orco olfactory co-receptor. iScience, 26(6), 106883.

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Mosquito Sperm Count and Analysis

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Three-to-five-day-old females were cold anesthetized and spermathecae preparation and sperm count was carried out as described in⁹⁶ (link) with modifications. Two pairs of fine tweezers were used to dissect spermathecae from each female on a slide (Color Frost Yellow, Fisher Scientific) with 50 μL of 1% phosphate buffered solution (PBS) under a dissecting microscope. Intact spermathecae from a female were transferred to 20 μL of PBS on a manually ruled slide, torn open using insect pins to release sperm and vigorously agitated to minimize sperm clumping. Insect pins used for each female’s spermathecal dissection were rinsed with 50 μL of PBS twice on the slide, after which the slide was dried at 65°C on a hot plate. Upon drying, sperm was fixed on slide with 70% ethanol at 65°C for 20 min. Vectashield Plus Antifade Mounting Medium with DAPI (Vector Laboratories) was applied to sperm on the slide for an hour. Sperm heads were viewed at 400x magnification under a flourescence microscope (DM5500B, Leica Microsystems) and counted by slowly walking across the ruled slide. Scoring was conducted by an observer who was blinded to the genotype of the sperm. This experiment was conducted across three batches of mosquitoes. For each batch, all genotypes were reared in parallel with the same feeding and environmental conditions.

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Spinal Cord Injury Histology

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Animals were anesthetized with an i.p. injection of overdosed Euthasol and perfused transcardially with 0.1 M PBS, pH 7.4, followed by 4% paraformaldehyde. The spinal cord was dissected, post-fixed overnight and cryoprotected in 30% sucrose in 0.1 M Tris-buffered saline (TBS). Spinal cord segment was trimmed to 1 cm containing the injury site in the center and was embedded in M1 matrix (Epredia)for cryosectioning. The spinal cord was serially sectioned in a longitudinal, horizontal plane at 35 μm in three series of sections mounted on the slides. For immunohistochemistry, sections were blocked in TBS with 0.25% Triton X and 5% donkey serum for 1 h, followed by incubation with primary antibodies for glial fibrillary acidic protein (GFAP, 1:1000, abcam) to label astrocytes, serotonin (5-HT, 1:1000, Immunostar) to label serotonergic axons, and collagen III (Col-III, 1:1000, abcam) to label collagen scars. Spinal cord sections were incubated with primary antibody overnight in 4 °C followed by incubation with AlexaFluor-488-, 594-, or 647-conjugated donkey secondary antibodies (1:500, Invitrogen). After thorough washing, the sections were coverslipped with mounting media. The slides were imaged using the fluorescent microscope (DM5500B, Leica Microsystems), connected to a digital camera (C11440, Hamamatsu), and installed with a Slidebook 6 software (Intelligent Imaging Innovations).

Fernandes S., Oatman E., Weinberger J., Dixon A., Osei-Owusu P, & Hou S. (2022). The susceptibility of cardiac arrhythmias after spinal cord crush injury in rats. Experimental neurology, 357, 114200.

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Lipid body staining in M. xanthus

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M. xanthus strains were grown to a density of 5×10⁸ cells/ml then resuspended in 100 µl dH₂O to a final density of 5×10⁹ cells/ml. Aliquots of 10 µl were spotted on TPM agar and incubated for various times. Lipid body staining was carried out as described by Hoiczyk et al with modifications [4] (link). A 0.5 mg/ml stock solution of Nile red (Sigma Aldrich) prepared in 100% ethanol was diluted in dH₂O to a final concentration of 1.25 µg/ml and added directly on top of cells developing on TPM agar. The plates were incubated for 2 h at 32°C. Cells were resuspended in a drop of TPMF buffer [TPM buffer containing 10% ficoll], and examined with a fluorescence microscope (Leica Microsystems, DM5500B). Digital images were obtained using a QICAM FAST 1394 camera (Q Imaging systems, Compix Inc.).
The fluorescence images were digitally altered using Simple PCI (Hamamatsu Corporation) to remove background noise, improve contrast, and measure cell length and fluorescence intensity. Average lipid body area and cell length were calculated from 30 randomly chosen cells.

Bhat S., Boynton T.O., Pham D, & Shimkets L.J. (2014). Fatty Acids from Membrane Lipids Become Incorporated into Lipid Bodies during Myxococcus xanthus Differentiation. PLoS ONE, 9(6), e99622.

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Quantifying Cell Proliferation with EdU Assay

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Cell proliferation was analyzed using a Click-iT™ EdU Imaging kit (cat. no. C10337; Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, cells were cultured at a density of 5x10⁴ cells/well (for 24 h propofol stimulation) or 3x10⁴ cells/well (for 48 h propofol stimulation) in 24-well plates for 24 h and then treated with different concentrations of propofol or 0.05% DMSO for 24 or 48 h. Following the incubation, the cells were labeled for 2 h with 10 µM EdU solution and then fixed with 4% paraformaldehyde (Shanghai Lingfeng Chemical Reagent Co., Ltd.) for 15 min at room temperature. Cells were permeabilized with 0.5% Triton X-100 (cat. no. 0694; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min. EdU-incorporated cells and all cells were stained with Alexa Fluor fluorescent dye and Hoechst 33342 solution, respectively, for 30 min at room temperature, away from light. Subsequently, stained cells were visualized in five randomly selected fields of view using a fluorescence microscope (cat. no. DM5500B; Leica Microsystems GmbH), and the percentage of EdU-positive cells was calculated. Analysis was performed using ImageJ version 1.8.0 software (National Institutes of Health).

Li C., Fu Q., Cai J., Mei H, & Shangguan W. (2021). Effects of propofol on the proliferation and migration of liver cancer cells. Experimental and Therapeutic Medicine, 22(1), 733.

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Fluorescent Nanoparticle Uptake in Caco-2 Cells

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Fluorescein-labeled NPs were formulated by the association of fluorescein isocyanate (FITC) with EA in PCL. The pigment was appended to the NPs by adding 100 mg FITC to 50 mg PCL dissolved in ethyl acetate, and FITC-EA-NPs were produced using the previous step as reported earlier in the preparation of EA-NPs.35 (link) After 24 h, the cumulative release of FITC from FITC-EA-NPs in PBS (pH =7.4) was assessed by spectrofluorometry (excitation/emission 494/512 nm) to ensure the existence of fluorescence during the experiment. Caco-2 cells were first propagated on 22 mm² sterile cover slips in six sterile microtiter well plates at 2×10⁵ cells/well in 1,000 μL of growth medium. Plates were incubated for 24 h before running the experiment. After seeding for 24 h, cells were exposed to fluorescent NPs or free EA for further 2 h. After the period of exposure, the attached cells to cover slips were thoroughly rinsed with PBS (pH =7.4) the attached cells were thoroughly rinsed thrice with PBS (pH =7.4) then placed for 5 min with 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI), which was used as counter stain to stain the nuclei, and inspected using an epifluorescence microscope (DM 5500 B; Leica Microsystems, Wetzlar, Germany) at different magnifications.

Mady F.M, & Shaker M.A. (2017). Enhanced anticancer activity and oral bioavailability of ellagic acid through encapsulation in biodegradable polymeric nanoparticles. International Journal of Nanomedicine, 12, 7405-7417.

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