Axiovision digital imaging system

Manufactured by Zeiss

Sourced in Germany

AxioVision is a digital imaging system from Zeiss. It is designed for capturing, processing, and analyzing digital images from a variety of microscopy techniques.

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12 protocols using axiovision digital imaging system

Characterization of BLPs-F and BLPs-H

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For indirect IFA, 100 µL of BLPs-F or BLPs-HN particles were resuspended in 3% bovine serum albumin (BSA) and blocked at 37°C for 30 min. Mouse anti His Tag monoclonal antibody (Beyotime Biotechnology, Shanghai, China) was used as the primary antibody and 1:500 diluted fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG was used as the secondary antibody. After the final wash, the particles were observed with a Zeiss microscope and the Zeiss Axiovision digital imaging system (Zeiss, Oberkochen, Germany).
For western blot analysis, the samples of BLPs-F and BLPs-H were separated by 10% SDS-PAGE under denaturing conditions and transferred onto polyvinylidene fluoride membranes to identify BLPs binding the recombinant proteins. The samples of sf9 cells infected with rBV-F-PA or rBV-H-PA served as the control. Immunoblot analysis was performed using a monoclonal antibody against His Tag. Immunodetection was carried out using a horseradish peroxide (HRP)-conjugated goat anti-mouse IgG and enhanced chemiluminescence.

Wang J., Liu L., Zong X., Wang C., Zhu G., Yang G., Jiang Y., Yang W., Huang H., Shi C., Zeng Y., Wang N., Cao X., Wang C, & Feng N. (2024). Immunogenicity and protective efficacy of a novel bacterium-like particle-based vaccine displaying canine distemper virus antigens in mice and dogs. Microbiology Spectrum, 12(4), e03477-23.

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SDS-PAGE and Western Blot of SUDV-GP1

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For the SDS-PAGE and WB analyses of the GEM particles, the eGP-PA-GEM complexes were treated with 5× SDS loading buffer for 10 min at 100 °C, separated using 10% SDS-PAGE gel, and then transferred onto a nitrocellulose (NC) membrane for WB analysis with the mouse anti-SUDV-GP1 monoclonal antibody. For IFA analysis, GEM particles with bound eGP-PA were blocked with 3% BSA for 30 min at 37 °C. Then, incubations with the primary antibody (mouse anti-SUDV-GP1 monoclonal antibody) and secondary antibody (FITC-labeled goat anti-mouse IgG) were performed as previously described (Section 2.2), and the particles were viewed and imaged using a Zeiss microscope with incident UV illumination and a Zeiss Axiovision digital imaging system (Zeiss, Oberkochen, Germany).

Xu S., Jiao C., Jin H., Li W., Li E., Cao Z., Shi Z., Yan F., Zhang S., He H., Chi H., Feng N., Zhao Y., Gao Y., Yang S., Wang J., Wang H, & Xia X. (2019). A Novel Bacterium-Like Particle-Based Vaccine Displaying the SUDV Glycoprotein Induces Potent Humoral and Cellular Immune Responses in Mice. Viruses, 11(12), 1149.

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Immunostaining and FISH Microscopy Protocol

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For immunostaining, cells were fixed with acetone for 10 min at 4 °C (Figures 1c and 3) or 4% HCHO for 20 min at room temperature (Figures 4d and e) or 30 °C (Figure 6), and HCHO-fixed cells were permeabilized in phosphate-buffered saline containing 0.5% Triton X-100 on ice. After blocking in phosphate-buffered saline containing 3% bovine serum albumin and 0.1% Tween-20, cells were stained with appropriate antibodies. Immunostained cells were viewed under an LSM710 confocal microscope with the Zen software (Carl Zeiss, Jena, Germany). The objective lenses were EC Plan-NEO FLUAR × 40/1.3 and Plan APOCHROMAT × 63/1.4. FISH was performed as described previously.^{9 (link), 10 (link)} Probes to the pericentromeric regions of chromosome 7, 8, 12 and 15 were purchased from Vysis (Downers Grove, IL, USA). FISH-stained cells were viewed under a Zeiss fluorescent microscope with a × 40/1.3 or a × 63/1.4 oil immersion objective and an AxioVision digital imaging system (Carl Zeiss).

Kuga T., Nie H., Kazami T., Satoh M., Matsushita K., Nomura F., Maeshima K., Nakayama Y, & Tomonaga T. (2014). Lamin B2 prevents chromosome instability by ensuring proper mitotic chromosome segregation. Oncogenesis, 3(3), e94-.

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Immunostaining of CD11b+ Cells in Tissue Sections

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For immunostaining, mounted tissue sections were fixed in acetone at 4°C for 10 min, washed in PBS for 5 min, and subsequently treated with 3% H₂O₂ for 5 min to reduce background staining. Excess H₂O₂ was removed by washing in PBS for 10 min. The sections were blocked with PBS containing 2% goat serum and 3% BSA for 1 h. Sections were then incubated at room temperature with FITC-labeled mouse anti-CD11b antibody (eBioscience) for 1 h, and then washed in PBS for 10 min. Positively stained cells were visualized under a Zeiss fluorescent microscope with an AxioVision digital imaging system (Carl Zeiss MicroImaging, Thornwood, NY).

Jiang X., Sato T., Yao Z., Keeney M., Pajarinen J., Lin T.H., Loi F., Egashira K., Goodman S, & Yang F. (2015). Local Delivery of Mutant CCL2 Protein-Reduced Orthopaedic Implant Wear Particle-Induced Osteolysis and Inflammation In Vivo. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 34(1), 58-64.

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Superoxide and Peroxynitrite Detection in Ischemia-Reperfusion

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We used hydroethidine (HEt) and HKYellow-AM fluorescent probes to detect superoxide and peroxynitrite in b.End3 cells, similar to in vivo detection. The b.End3 cells were incubated with HEt and HKYellow-AM (10 µM) separately at 37 °C for 20 min after 5 h of OGD plus 5 h of reoxygenation, with or without t-PA treatment or AGNHW treatment. A fluorescence microscope (Carl Zeiss) with an Axio Vision digital imaging system was used to detect the fluorescent signals.

Chen H., Luo Y., Tsoi B., Gu B., Qi S, & Shen J. (2022). Angong Niuhuang Wan reduces hemorrhagic transformation and mortality in ischemic stroke rats with delayed thrombolysis: involvement of peroxynitrite-mediated MMP-9 activation. Chinese Medicine, 17, 51.

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Microscopic Imaging Techniques for Fluorescence Analysis

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All slides were analyzed using a Zeiss Axio Imager.M2 microscope equipped with an Axiovision digital imaging system (Zeiss) using 20× or 40× objectives or a Zeiss LSM 880 Axio observer confocal microscope with Airyscan using a 63× objective (Zeiss). The 20× objective for the Axio imager was a Plan-apochromat with a numerical aperture of 0.8 M27. The 40× objective for the Axio imager was a Plan-apochromat with a numerical aperture of 0.95 Korr M27. The 63× objective for the Zeiss LSM 880 confocal microscope was a Plan-apochromat oil-immersion objective for Airyscan with a numerical aperture of 1.4. Fluorescent donkey secondary antibodies were used that were conjugated to Alexa Fluor 488, Cy3, Cy5, or Alexa Fluor 796. Imaging of fixed tissue was performed at room temperature; all specimens were mounted in ProLong Gold antifade reagent before imaging. The camera used to acquire images on the Axio Imager microscope was the AxioCam HRm Rev.3. For images acquired using the Zeiss LSM 880 Airyscan, superresolution processing was used. Axiovision digital imaging system and Zen blue software were used to export single-channel tiff images for all images acquired. Adobe Photoshop was used to merge images.

Engevik A.C., Kaji I., Postema M.M., Faust J.J., Meyer A.R., Williams J.A., Fitz G.N., Tyska M.J., Wilson J.M, & Goldenring J.R. (2019). Loss of myosin Vb promotes apical bulk endocytosis in neonatal enterocytes. The Journal of Cell Biology, 218(11), 3647-3662.

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Quantifying ONOO- Production In Vivo and In Vitro

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HKYellow-AM is our newly developed ONOO⁻ fluorescent probe with high sensitivity and selectivity (Gong et al., 2015 (link); Peng et al., 2016 (link)). We detected ONOO⁻ production both in vivo and in vitro experiments. For in vivo study, the mice at 18 dpi were intravenously injected with HKYellow-AM (10 μM, 1 mL/kg) at 15 min before sacrificed. After perfused with PBS, the fresh L4-L6 spinal cords were immediately dissected, embedded into O.T.C., cut into 30 μm sections, counterstained the nucleus with DAPI and imaged by a confocal laser scanning microscope LSM 780 at the conditions of excitation wavelength of 543 nm and emission wavelength of 567 nm. For in vitro study, cells were stained with 10 μM HKYellow-AM for 30 min and washed with PBS. The fluorescent images were captured by Carl Zeiss fluorescent microscope equipped with Axio Vision digital imaging system.

Li W., Wu H., Gao C., Yang D., Yang D, & Shen J. (2018). Radix Rehmanniae Extract Ameliorates Experimental Autoimmune Encephalomyelitis by Suppressing Macrophage-Derived Nitrative Damage. Frontiers in Physiology, 9, 864.

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Immunofluorescence Staining of Duodenal Tissues

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Normal human duodenum sections and those from the patient with the DGAT1 mutation were deparaffinized and were submitted to antigen retrieval in a pressure cooker using the target retrieval solution (Dako North America Inc.). Serum-free protein block (Dako North America Inc.) was used for blocking the tisssue, and immunofluorescence staining was performed. In short, tissue sections were incubated overnight at 4°C with primary antibodies diluted in Antibody Diluent with Background Reducing Components (Dako North America Inc.). Appropriate secondary antibodies were conjugated for immunofluorescence with Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 647, Cy3, or Cy5 (1-hour incubation at room temperature). Primary antibodies are listed in Table 1. Tissue sections were washed 3 times in PBS and mounted with ProLong Gold plus DAPI (Invitrogen). All imaging was performed using the Olympus FV-1000 or Zeiss Axiophot microscope equipped with an Axiovision digital imaging system (Zeiss, Jena GmBH, Germany).

Schlegel C., Lapierre L.A., Weis V.G., Williams J.A., Kaji I., Pinzon-Guzman C., Prasad N., Boone B., Jones A., Correa H., Levy S.E., Han X., Wang M., Thomsen K., Acra S, & Goldenring J.R. (2018). Reversible deficits in apical transporter trafficking associated with DGAT1 deficiency. Traffic (Copenhagen, Denmark), 19(11), 879-892.

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TUNEL Assay for Apoptosis Detection

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Apoptotic cell death was determined by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Briefly, rat brain samples were fixed with 4% paraformaldehyde (PFA) and then immersed in 30% sucrose until it sank. Samples were then embedded in O.C.T. and cut into a section of 25 μm. TUNEL staining was conducted referring to the manufacturer’s instructions in the TUNEL assay kit (Shanghai YEASEN Biotechnology Co.). Hoechst staining was used to visualize the cell nucleus. A fluorescence microscope (Carl Zeiss) with Axio Vision digital imaging system was applied to obtain the fluorescence images.

Luo Y., Chen H., Tsoi B., Wang Q, & Shen J. (2021). Danggui-Shaoyao-San (DSS) Ameliorates Cerebral Ischemia-Reperfusion Injury via Activating SIRT1 Signaling and Inhibiting NADPH Oxidases. Frontiers in Pharmacology, 12, 653795.

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Immunostaining Imaging Protocols

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All immunostaining was analyzed using a Zeiss Axio Imager.M2 microscope equipped with an Axiovision digital imaging system (Zeiss, Oberkochen, Germany) using a 20× objective or a Nikon Ti-E microscope with an A1R laser scanning confocal system (Nikon Instruments, Inc, Melville, NY) using a 60× objective. The Axio imager 20× objective has a Plan-apochromat with a numeric aperture of 0.8 M27. The Nikon A1R 60× oil objective has a Plan apochromat with a numeric aperture of 1.4. Imaging of tissue sections was performed at room temperature and all tissue sections were mounted in ProLong Gold Antifade reagent. The Axio Imager microscope was equipped with an AxioCam HRm Rev.3 camera. The Axiovision digital imaging system was used to export single-channel grayscale TIFF images for all images acquired on the Axio Imager. The Nikon A1R used a Nikon A1 plus camera. Nikon Elements software was used to export images as individual channel TIFF files. Adobe Photoshop (Adobe, San Jose, CA) was used to merge individual channel images into Red Green Blue (RGB).

Engevik A.C., Krystofiak E.S., Kaji I., Meyer A.R., Weis V.G., Goldstein A., Coutts A.W., Melkamu T., Saqui-Salces M, & Goldenring J.R. (2021). Recruitment of Polarity Complexes and Tight Junction Proteins to the Site of Apical Bulk Endocytosis. Cellular and Molecular Gastroenterology and Hepatology, 12(1), 59-80.

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