Axioskop2 mot plus microscope

Manufactured by Zeiss

Sourced in Germany, Switzerland, United States

The Axioskop2 mot plus is a motorized microscope designed for versatile applications in research and clinical laboratories. It features a robust and ergonomic design, allowing for smooth and precise control of the motorized components. The microscope is equipped with a range of optical components, providing high-quality imaging capabilities.

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Axioskop2 MOT Plus Wide Field Fluorescence Microscope (6 citations) Axioskop2 MOT Plus Apotome microscope (6 citations)

76 protocols using axioskop2 mot plus microscope

Histological Analysis of Kidney and Mesentery

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The kidney and mesenteric samples were fixed in 4% buffered formalin for 24 hours, dehydrated in graded ethanol and embedded in paraffin for immunohistochemical and histopathological analysis.
Kidneys: Sections of 4 μm were stained with hematoxylin–eosin and analyzed using a high-power objective (40X) on an Axioskop 2-Mot Plus Zeiss microscope (Carl Zeiss, Jena, Germany).
Mesentery: The mesenteric fragments were also fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, sodium cacodylate buffer 0.1 M (pH 7.4) for 24 h at 4°C, dehydrated by graded methanol, and embedded in LR Gold resin (London Resin; Reading, Berkshire, U.K.) for the quantitative analysis of the neutrophils and mast cells. The mesenteric infiltrating cells were evaluated in 1 μm tissue sections embedded in LRGold and counted using a high-power objective (40x) on an Axioskop 2-Mot Plus Zeiss microscope (Carl Zeiss, Jena, Germany). The average number of neutrophils and mast cells was calculated and recorded. The values were reported as the mean ±SEM of the number of cells/mm². Mast cells were also analyzed morphologically and classified as either intact or degranulated after staining with toluidine blue 0.5% (Taab Laboratories, UK).

Stuqui B., de Paula-Silva M., Carlos C.P., Ullah A., Arni R.K., Gil C.D, & Oliani S.M. (2015). Ac2-26 Mimetic Peptide of Annexin A1 Inhibits Local and Systemic Inflammatory Processes Induced by Bothrops moojeni Venom and the Lys-49 Phospholipase A2 in a Rat Model. PLoS ONE, 10(7), e0130803.

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Immunofluorescent Detection of Antigen Expression

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Antigen expression was confirmed on ice-cold acetone fixed 8 μm cryostat sections of SKRC52 and CT26-CAIX stained with IL2-XE114-TNF^mut and IL2-F8-TNF^mut (final concentration 5 μg/mL) and detected with rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. IL2-KSF-TNF^mut (specific for an irrelevant antigen) was used as negative control. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).
For ex vivo immunofluorescence analysis, mice were injected with 50–60 μg IL2-XE114-TNF^mut, IL2-F8-TNF^mut, or IL2-KSF-TNF^mut and sacrificed 24 h after injection. Organs were excised and embedded in cryo-embedding medium (Thermo Scientific) and cryostat section (10 μm) were stained using the following antibodies: rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).

De Luca R., Gouyou B., Ongaro T., Villa A., Ziffels B., Sannino A., Buttinoni G., Galeazzi S., Mazzacuva M, & Neri D. (2019). A Novel Fully-Human Potency-Matched Dual Cytokine-Antibody Fusion Protein Targets Carbonic Anhydrase IX in Renal Cell Carcinomas. Frontiers in Oncology, 9, 1228.

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Antigen Expression and Tumor Targeting

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Antigen expression was confirmed on ice-cold acetone fixed 8 μm cryostat sections of F9, C51 and WEHI-164 stained with F8-F8-IL15, F8-F8-SD-IL15, KSF-KSF-IL15 and KSF-KSF-SD-IL15 (final concentration 0.01mg/mL) and detected with Protein A-AlexaFluor488 (Invitrogen P11047). A rat anti-mouse CD31 (BD 550274) was used in order to detect blood vessels and detected with donkey anti-rat ALEXA 594 (Invitrogen A21209). Slides were mounted with fluorescent mounting medium containing DAPI (Dako Omnins GM304) and analyzed with Axioskop2 mot plus microscope (Zeiss). For ex vivo immunofluorescence analysis, 129/SvEv mice bearing F9 teratocarcinomas were injected intravenously with 100 μg of F8-F8-IL15, F8-F8-SD-IL15, KSF-KSF-IL15 and KSF-KSF-SD-IL15. After 24 hours mice were sacrificed, tumors and organs were excised and embedded in cryo-embedding medium (Thermo Scientific). Cryostat section of 8 μm were stained with Protein A-AlexaFluor488 (Invitrogen P11047) and donkey anti-rat ALEXA 594 in order to detect the blood vessels stained with rat anti-mouse CD31 (BD 550274). Slides were mounted with fluorescent mounting medium containing DAPI (Dako Omnins GM304) and analyzed with Axioskop2 mot plus microscope (Zeiss).

Corbellari R., Stringhini M., Mock J., Ongaro T., Villa A., Neri D, & De Luca R. (2021). A novel antibody-interleukin-15 fusion protein selectively localizes to tumors, synergizes with TNF-based immunocytokine and inhibits metastasis. Molecular cancer therapeutics, 20(5), 859-871.

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Immunofluorescent Characterization of Cell Types

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Cells from PVR membranes were seeded on a four-well Chamber Slide System for 24 to 72 hours (Thermo Fisher Scientific, Waltham, MA, USA). Cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes, washed with PBS, permeabilized with 0.25% Triton X-100 in PBS for 5 minutes, and blocked (10% goat serum in PBS) for 1 hour. To identify specific cell types, C-PVR was incubated with a fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-SMA primary antibody (smooth muscle actin, 1:150; Sigma-Aldrich Corp.), rabbit polyclonal anti-GFAP primary antibody (glial fibrillary acidic protein, 1:150; Dako), rabbit anti-F4/80 antibody (1:200; Abcam), and rabbit anti-cytokeratin antibody (1:200; Abcam). Cells were incubated with the primary antibody overnight at 4°C followed by incubation with goat anti-rabbit Alexa Fluor 594 secondary antibody (1:300; Life Technologies) or with goat anti-mouse Alexa Fluor 488 secondary antibody (1:300; Life Technologies) for 2 hours at room temperature. Cells were washed with PBS three times, and coverslips were mounted onto the chamber slides using a Prolong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies). Images were obtained using a Zeiss Axioskop 2 MOT Plus microscope (Carl Zeiss, Inc., Oberkochen, Germany).

Amarnani D., Machuca-Parra A.I., Wong L.L., Marko C.K., Stefater J.A., Stryjewski T.P., Eliott D., Arboleda-Velasquez J.F, & Kim L.A. (2017). Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy. Investigative Ophthalmology & Visual Science, 58(10), 3940-3949.

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Cryptococcus neoformans Capsule Analysis

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O/N cultures of C. neoformans were collected by centrifugation, washed twice with sterile PBS, diluted to 10⁶ cells/mL in DMEM and incubated at 37°C in 5% CO₂ for 24 h in T-75 tissue culture flasks or 24-well plates. The cells were then washed and resuspended in PBS, mixed with 1.5 parts India Ink, and viewed by light microscopy with a ZEISS Axioskop2 MOT Plus microscope (Carl Zeiss Microscopy, LLC).
For antibody detection of cell wall-associated GXM, strains were induced as above for 24 h, fixed for 1 h in 3.7% formaldehyde, washed in PBS, and then incubated for 1 h at room temperature (RT) with 1 mg/mL of anti-GXM monoclonal antibody (mAb) F12D2 or 302 (from Dr. Thomas R. Kozel, University of Nevada School of Medicine) conjugated to AlexaFlour 488. Stained cells were washed twice with PBS, resuspended in PBS, and examined on a ZEISS Axioskop 2 MOT Plus microscope.

Li L.X., Rautengarten C., Heazlewood J.L, & Doering T.L. (2018). Xylose donor transport is critical for fungal virulence. PLoS Pathogens, 14(1), e1006765.

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Microscopy Techniques for Cell Imaging

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An inverted widefield Nikon Ti Eclipse microscope, Andor NeoZyla camera and Nikon Elements v4.60 acquisition software was used with a 60× (NA 1.40 PlanApo) oil objective for immunofluorescent cell imaging in micropattern, Blebbistatin, area, and stretch experiments and with a 10× (NA 0.45, PlanApo) objective for motility experiments. Area measurements were performed with Nikon Elements Analysis v4.6 software (binary auto-detect to ROI measurements) with 60× images calibrated at 0.11 µm/px. An upright widefield Zeiss Axioskop2 mot plus microscope (40× NeoFluor 0.75NA dry objective) was used with Zeiss AxioCamMRm camera and Zeiss AxioVision v4.8.1 software. Confocal images were acquired on a Leica SP8 DMi8 inverted microscope (Leica 63× oil objective NA 1.40 HC PL APO CS2) with PMT and hybrid detectors and LASX v3.5.7 software. Images are presented as maximum intensity projections of a confocal stack of Lightning deconvolved images. Leica acquisition was used for stretch, micropattern, Blebbistatin, and retrograde flow experiments.

Hoffman L.M., Jensen C.C, & Beckerle M.C. (2022). Phosphorylation of the small heat shock protein HspB1 regulates cytoskeletal recruitment and cell motility. Molecular Biology of the Cell, 33(11), ar100.

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Methotrexate Inhibits Proliferation of C-PVR Cells

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C-PVR cells were cultured on cover slips at a confluency of 30,000 cells per well and treated with different concentrations of MTX (100, 200, and 400 μM) or vehicle. At the 24-, 48-, and 72-hour time points, cells were washed and fixed with 4% PFA for 10 minutes. Cells were permeabilized with 0.5% Triton X-100 in PBS for 5 minutes and blocked (10% goat serum in PBS) for 1 hour at room temperature. Proliferation was evaluated by incubating cells with anti-Ki67 antibody (1:50; Novus Biologicals, Cambridge, United Kingdom). The primary antibody was prepared in antibody dilution buffer (5% goat serum) and incubated overnight at 4°C. Cells were incubated with goat anti-rabbit Alexa Fluor 594 secondary antibody (Life Technologies) at 1:300 dilution for 2 hours at room temperature. The cover slips were washed and mounted onto slides using Prolong Gold Antifade Reagent with DAPI. Images were obtained using a Zeiss Axioskop 2 MOT Plus microscope (Carl Zeiss, Inc.). The quantification is reported as an average of Ki67-positive cells to the number of DAPI-positive cells per high-powered field of human C-PVR cultures, which were cultured in triplicate.

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Multimodal Immunofluorescence Imaging of Vascular Cells

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C-FVMs were seeded on a four-well Chamber Slide System (Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X-100 in PBS for 5 min, and blocked (10% goat serum in PBS) for 1 h. To identify smooth muscle cells and myofibroblasts, cultures were incubated with a fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-SMA primary antibody (1:150; Sigma). To label astrocytes and glial cells, cells were incubated with a rabbit polyclonal anti-GFAP primary antibody (1:150; Dako) overnight at 4 °C followed by incubation with goat anti-rabbit Alexa Fluor 488 secondary antibody (1:200; Life Technologies, Carlsbad, CA) for 2 h at room temperature. CD31 staining was performed using a similar procedure with the CD31 primary antibody (1:50; Dako, Carpinteria, CA) and anti-mouse Alexa Fluor 647 secondary antibody (1:200; Life Technologies, Carlsbad, CA). Isolectin B4 staining was performed using isolectin B4 primary antibody directly conjugated to Alexa Fluor 648 (1:100, Invitrogen, Carlsbad, CA). Coverslips were mounted onto the chamber slides using a Prolong Gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI; Life Technologies). Images were obtained using a Zeiss Axioskop 2 MOT Plus microscope (Carl Zeiss Inc.).

Kim L.A., Wong L.L., Amarnani D.S., Bigger-Allen A.A., Hu Y., Marko C.K., Eliott D., Shah V.A., McGuone D., Stemmer-Rachamimov A.O., Gai X., D’Amore P.A, & Arboleda-Velasquez J.F. (2015). Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy. Molecular Vision, 21, 673-687.

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Quantification of Microbial Cell Abundances

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Samples were taken from the initial microbial inocula (day 0) and all microcosms (day 5) for determinations of microbial cell abundances. For bacterial abundance, 4 mL were preserved with formaldehyde at a final concentration of 2%, and immediately flash frozen in liquid nitrogen until used in the flow cytometry measurement. Cells in the samples were stained using 10x SYBR Green I (Life Technologies, Darmstadt, Germany) and then counted in a FASCalibur flow cytometer (Becton Dickinson, Fremont, CA, United States), as described elsewhere (Gasol and del Giorgio, 2000 (link)).
In addition, the cell abundance of protists was determined using epifluorescence microscopy, according to Weber et al. (2012) (link) with minor modifications. Briefly, 10 mL of sample was fixed with formaldehyde at a final concentration of 2% and stored at 4°C. After ∼4 h, the fixed samples were filtered onto 0.8-μm, 25-mm black filters (Whatman, Dassel, Germany), which were then stored at −20°C until further processing. For cell enumeration, cells on the filters were stained with 4’,6-diamidin-2-phenylindol and three randomly selected fields of view were inspected at 63× magnification using a Zeiss Axioskop 2 mot plus microscope (Zeiss, Oberkochen, Germany). Technical triplicates were established for each sample.

Shen D., Langenheder S, & Jürgens K. (2018). Dispersal Modifies the Diversity and Composition of Active Bacterial Communities in Response to a Salinity Disturbance. Frontiers in Microbiology, 9, 2188.

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Capsule Induction in Cryptococcus neoformans

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Cultures of C. neoformans grown in YPD O/N were collected by centrifugation (3,000 × g, 5 min) and washed twice with sterile PBS. The cells were then resuspended in DMEM at 10⁶ cells/ml in T-75 tissue culture flasks or 24-well plates and incubated at 37°C with 5% CO₂ for 24 h to induce production of capsule. Induced cells were collected, washed, and resuspended in PBS, mixed with 1.5 parts India ink (Chartpak, Inc.), and viewed with a Zeiss Axioskop2 MOT Plus microscope (Carl Zeiss Microscopy, LLC).

Li L.X., Rautengarten C., Heazlewood J.L, & Doering T.L. (2018). UDP-Glucuronic Acid Transport Is Required for Virulence of Cryptococcus neoformans. mBio, 9(1), e02319-17.

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