Af6500

Manufactured by Leica

Sourced in Germany

The Leica AF6500 is a high-performance automated fluorescence microscope designed for a wide range of applications. It features a motorized stage, autofocus system, and advanced imaging capabilities to provide precise and reproducible results.

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Lab products found in correlation

Las af software, by Leica (3 mentions) Dmi6000b microscope, by Leica (2 mentions) Du8285 vp, by Oxford Instruments (2 mentions) Lsm 710 laser scanning microscope, by Zeiss (1 mentions) Rabbit anti ki67, by Abcam (1 mentions) Rabbit anti vegf, by Abcam (1 mentions) Rabbit anti somatostatin, by Abcam (1 mentions) Goat anti pancreatic polypeptide, by Abcam (1 mentions) Vb 6000, by Keyence (1 mentions) Fv10 asw software, by Olympus (1 mentions) Deltavision personal system, by Cytiva (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions) Uplansapo 100x 1, by Olympus (1 mentions) Facscanto 2, by BD (1 mentions) Tcs sp, by Leica (1 mentions) Lsm 780, by Zeiss (1 mentions)

Spelling variants of this product

Leica AF6500 Integrated System for Imaging and Analysis (11 citations) Leica AF6500 microscope (2 citations)

10 protocols using af6500

Comprehensive Histological and Immunohistochemical Analysis of Mouse Tissues

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Images of the mice and their organs were obtained using a digital fluorescent microscope (VB-6000; Keyence Japan, Osaka, Japan, or Leica AF6500; Leica Microsystems, Tokyo, Japan). For histological analyses, tissues were fixed in 10% formalin solution and embedded in paraffin for hematoxylin and eosin (H&E) staining. Alternatively, tissues were fixed by perfusion with 4% paraformaldehyde. Frozen sections were the cut using a Microm HM500 OM Microtome Cryostat (Carl Zeiss Japan, Tokyo, Japan).
The following primary antibodies were used for immunohistochemical analysis: guinea pig anti-insulin, rabbit anti-glucagon, rabbit anti-somatostatin, goat anti-pancreatic polypeptide, rabbit anti-chromograninA (cgA), rabbit anti-Ki67, and rabbit anti-VEGF (Abcam, Tokyo, Japan). Alexa568- or Cy3- labeled species-specific anti-IgG antibodies (Life Technologies Japan, Tokyo, Japan) were used as secondary antibodies. Images were obtained using a LSM710 laser scanning microscope (Carl Zeiss Japan), or a HS BZ-9000 fluorescent microscope system (Keyence). The Ki-67 index was calculated by dividing the total number of nuclei by the number of Ki-67-positive nuclei. The number of nuclei was counted by observing 8–10 fields with a 40× lens.

Takano Y., Kasai K., Takagishi Y., Kikumori T., Imai T., Murata Y, & Hayashi Y. (2015). Pancreatic Neuroendocrine Tumors in Mice Deficient in Proglucagon-Derived Peptides. PLoS ONE, 10(7), e0133812.

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Fluorescence Imaging of Autophagy Markers

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Cells expressing GFP-Atg8 or GFP-Atg1 were observed under a FLUOVIEW FV1000 and edited using the Olympus FV10-ASW software (Olympus, Tokyo, Japan). For visualization of IM and Atg9-3xGFP, yeast cells were observed on a Leica AF6500 fluorescence imaging system (Leica Microsystems) mounted on a DMI6000 B microscope (HCX PL APO 100/NA=1.40-0.70, oil-immersion objective lens, xenon lamp; Leica Microsystems) under the control of the LAS-AF software (Leica Microsystems).

Ogasawara Y., Kira S., Mukai Y., Noda T, & Yamamoto A. (2016). Ole1, fatty acid desaturase, is required for Atg9 delivery and isolation membrane expansion during autophagy in Saccharomyces cerevisiae. Biology Open, 6(1), 35-40.

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Polysome Profile Analysis of Vanillin

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Vanillin and dimethyl sulfoxide (DMSO) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Stock solutions of 2 M vanillin were prepared in DMSO and stored at –30°C. Exponentially growing cells were harvested at OD₆₀₀ = 0.5 and treated with varying concentrations of vanillin. Polysome profile analysis was performed using the method described by Inada and Aiba (2005) (link). Polysome ratio was determined as the percentage of the area under polysomal ribosome peaks relative to the area under total ribosome peaks, according to the method of Hofmann et al. (2012) (link). A Leica AF6500 (Leica Microsystems, Wetzlar, Germany) fluorescence microscopic system was used for analysis. Cells treated with vanillin were observed immediately after treatment without fixation. The concentrations of vanillin in the culture medium were measured by high performance liquid chromatography (HPLC) as described in a previous report (Nguyen et al., 2014b (link)).

Nguyen T.T., Iwaki A, & Izawa S. (2015). The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress. Frontiers in Microbiology, 6, 1390.

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Imaging of Nitrogen Starvation Response

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Cells were grown in SCD or YMM medium containing ammonium sulfate as the sole nitrogen source, and then shifted to nitrogen-free YMM for 30 minutes, after which the indicated amino acids were added. Cells were collected by centrifugation (600 x g, 2 min) and subjected to microscopy. The cells were observed on a Leica AF6500 fluorescence imaging system (Leica Microsystems) mounted on a DMI6000B microscope (HCX PL APO 100/1.40–0.70 oil-immersion objective lens, xenon lamp (Leica Microsystems)) under the control of the LAS-AF software (Leica Microsystems). For time-lapse imaging, the cells were grown in YMM medium containing ammonium sulfate as the sole nitrogen source, and then shifted to nitrogen-free YMM on a glass bottom dish (Matsunami Glass) mounted with 2 mg/ml of concanavalin A (Sigma). Cells were then subjected to time-lapse imaging after addition of glutamine (final concentration of 0.5 mg/ml). Images were recorded using a DeltaVision Personal system (Applied Precision) mounted on a IX71 microscope (UPlanSApo 100x/1.40 oil-immersion objective lens, LED lamp (OLYMPUS)). ImageJ software (National Institutes of Health) was used to process and produce merged images.

Ukai H., Araki Y., Kira S., Oikawa Y., May A.I, & Noda T. (2018). Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane. PLoS Genetics, 14(4), e1007334.

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Vacuolar Membrane Labeling in Yeast

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Cells grown in SCD were collected with centrifugation (600 × g, 2 min) and subjected to microscopy. For FM4-64 staining, cells were pelleted and resuspended in 100 μl of culture medium containing 1 μl of 1.64 mM FM4-64, incubated for 30 min, washed twice with 1 ml SCD medium, incubated for 1 h, and then subjected to microscopy. Yeast cells were observed on a Leica AF6500 fluorescence imaging system (Leica Microsystems) mounted on a DIM6000 B microscope (HCX PL APO 63.6/1.40–0.60 oil-immersion objective lens, xenon lamp; Leica Microsystems, Wetzlar, Germany) under the control of the LAS-AF software (Leica Microsystems). Line-plot analysis of GFP signal intensities was conducted with ImageJ software (National Institutes of Health, Bethesda, MD) by the following procedure: designated lines were set across single vacuole, avoiding perivacuolar puncta. The intensity of GFP at a pixel corresponding to FM4-64 peak signal intensities was subtracted from intensities of the background region over ≥10 pixels.

Kira S., Kumano Y., Ukai H., Takeda E., Matsuura A, & Noda T. (2016). Dynamic relocation of the TORC1–Gtr1/2–Ego1/2/3 complex is regulated by Gtr1 and Gtr2. Molecular Biology of the Cell, 27(2), 382-396.

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Real-Time Monitoring of GPCR Internalization

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Flp-In T-REx HEK293 permanently expressing MOP-YFP receptors and treated for 24 hours with doxycycline [0.01 μg/ml] to induce the expression of c-myc-5-HT_2C-Cerulean receptors were grown on poly-d-lysine-treated coverslips. The coverslips were placed into a microscope chamber containing physiological saline solution (130 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 20 mM HEPES, and 10 mM d-glucose, pH 7.4). For internalization experiments in real time, drugs diluted in physiological saline solution were perfused into the microscope chamber and ≈7 m thick Z-stacks (15 planes of 0.49 m Z-step size) were acquired for 15 minutes at a rate of 1 frame per minute in an inverted epifluorescence microscope (AF6500; Leica Microsystems) equipped with temperature control (PeCon GmbH) at 37ºC. Cell fluorescence were visualized with a 63x 1.3NA oil objective, 470/40 excitation and 515LP emission filters, and images (1004x1002 pixels; 0.13m pixel size) were acquired with a DU8285_VP (Andor Technology) camera using exposures of 200ms or less and an EM Gain of 20.

Campa V.M., Capilla A., Varela M.J., de la Rocha A.M., Fernandez-Troyano J.C., Barreiro R.B, & Lopez-Gimenez J.F. (2015). Endocytosis as a Biological Response in Receptor Pharmacology: Evaluation by Fluorescence Microscopy. PLoS ONE, 10(4), e0122604.

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Quantifying Chondrocyte Oxidative Stress

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Cultured chondrocytes were washed with PBS and trypsinized for 5 min at 37 °C under 20% O₂ condition. FBS-free medium was added and trypsinized cells were collected and centrifuged for 5 min, 200 x g to isolate chondrocytes. The cells were stained and incubated for 30 min at 37 °C with 10 μM DHE in culture medium. Then, the cell suspension was washed with PBS, and staining was visualized using a BD FACS Canto II flow cytometer.
Tamoxifen-inducible Sod2 deficient chondrocytes were stained for 30 min at 37 °C with 10 μM DHE. After staining, the culture dishes were washed twice using PBS and then re-suspended in PBS. The fluorescence image was evaluated by a microfluorescence system (AF6500; Leica Microsystems, Wetzlar, Germany) and captured using the AF6500 software program.

Koike M., Nojiri H., Ozawa Y., Watanabe K., Muramatsu Y., Kaneko H., Morikawa D., Kobayashi K., Saita Y., Sasho T., Shirasawa T., Yokote K., Kaneko K, & Shimizu T. (2015). Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration. Scientific Reports, 5, 11722.

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Real-Time Imaging of GPCR Internalization

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Flp-In T-REx HEK293 cells permanently expressing MOP-YFP receptors and treated at different time points from 24 hours to 120 hours with doxycycline (10 ng.mL -1 ) to induce the expression of FLAG-M3-Cerulean receptors were grown on poly-d-lysine-treated coverslips.
Coverslips were placed into a microscope chamber containing physiological saline solution (130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1mM MgCl2, 20 mM HEPES, and 10 mM Dglucose, pH 7.4). To conduct internalisation experiments in real time, drugs were diluted in physiological saline solution and perfused into the microscope chamber and 15 planes of 0.49 m Z-step size were acquired for 15 minutes at a rate of 1 frame per minute in an inverted epifluorescence microscope (AF6500; Leica Microsystems) equipped with temperature control (PeCon GmbH) at 37 C. Cell fluorescence was visualized with a 63x 1.3 NA oil objective, 470/40 excitation and 515LP emission filters, and images (1004x1002 pixels; 0.13 m pixel size) were acquired with a DU8285_VP (Andor Technology) camera using exposures of 200 ms or less and an EM Gain of 20. Images were subsequently analysed with the Q_Endosomes algorithm previously described [14] by using ImageJ and Matlab software.

Lopez-Gimenez J.F., Alvarez-Curto E, & Milligan G. (2017). M3 muscarinic acetylcholine receptor facilitates the endocytosis of mu opioid receptor mediated by morphine independently of the formation of heteromeric complexes. Cellular signalling, 35.

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Visualizing Stress Granule Formation in Virus-Infected Cells

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Virus-infected HeLa or HeLa/G-G3BP cells were fixed with 4% paraformaldehyde (PFA) for 20 min at 4°C, permeabilized with 0.05% Triton X-100 in PBS for 5 min at room temperature (RT), blocked with 5 mg/ml BSA in PBST (0.04% Tween20 in PBS) for 30 min, and incubated at 4°C overnight with the relevant primary antibodies diluted in blocking buffer. The cells were then incubated with secondary antibodies at room temperature for 1 h. Nuclei were stained with 4.6-dimaidino-2-phenylinodole (DAPI) and analyzed with a confocal laser microscope, TCS-SP (Leica).
For fluorescence live imaging, HeLa/G-G3BP cells were stimulated by either NDV infection or RNA ligand transfection as described above. After 12 h stimulation, GFP fluorescence images were taken and analyzed with a fluorescence microscope system, AF6500 (Leica). The percentages of avSG-containing cells were calculated in more than five randomly chosen fields for each slide.

Yoo J.S., Takahasi K., Ng C.S., Ouda R., Onomoto K., Yoneyama M., Lai J.C., Lattmann S., Nagamine Y., Matsui T., Iwabuchi K., Kato H, & Fujita T. (2014). DHX36 Enhances RIG-I Signaling by Facilitating PKR-Mediated Antiviral Stress Granule Formation. PLoS Pathogens, 10(3), e1004012.

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Dyrk1a Genotype and Brain Tissue Analysis

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To obtain brain tissue, P7 and adult Dyrk1a^+/+ and Dyrk1a^+/− mice (4-9 animals each condition) were deeply anesthetized and transcardially perfused with 4% paraformaldehyde. The mouse’s brain was removed and post-fixed, and cryotome or vibratome brain sections were immunostained as described previously (Barallobre et al., 2014 (link)). To quantify neurons and synapses, images were obtained on a Leica AF6500 motorized wide-field microscope and a Zeiss LSM780 confocal microscope, respectively. To quantify BrdU-labeled cells, images were obtained on an Olympus BX51 motorized microscope with a JVC digital color camera. The procedures for immunostaining, and cell and synapse quantification are indicated in the Supplementary Materials and Methods. The source of the primary antibodies for these procedures is indicated in Supplementary Table 6.

Arranz J., Balducci E., Arató K., Sánchez-Elexpuru G., Najas S., Parras A., Rebollo E., Pijuan I., Erb L., Verde G., Sahun I., Barallobre M.J., Lucas J.J., Sánchez M.P., de la Luna S, & Arbonés M.L. (2019). Impaired development of neocortical circuits contributes to the neurological alterations in DYRK1A haploinsufficiency syndrome. Neurobiology of disease, 127, 210-222.

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