After 2 days of in vitro culture, chimeric organoids were fixed in 4% PFA for 10 min, permeabilized with 100% cold methanol for 10 min, and incubated with mouse anti-E-cadherin (1:100, BD Biosciences), rabbit anti-WT1 (1:50, Santa Cruz Biotechnology) followed by species-specific secondary antibodies (1:50; Jackson ImmunoResearch Laboratories). To detect human cells, chimeric organoids were incubated with FITC-conjugated anti–Human Nuclear Antigen (HNA, 1:100; Merck Millipore Ltd). Chimeric organoids were mounted with Dako Fluorescence Mounting Medium (DAKO Corporation) and examined using an inverted confocal laser-scanning microscope (LS 510 Meta; Carl Zeiss). 3D images of chimeric organoids were reconstructed using the Axio Vision Imager software (Carl Zeiss).
Axio vision imager
Manufactured by Zeiss
Sourced in Germany
The Axio Vision Imager is a high-performance laboratory imaging system designed for a wide range of microscopy applications. It offers advanced imaging capabilities, including high-resolution image capture, flexible image processing, and intuitive software control.
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Lab products found in correlation
2 protocols using axio vision imager
1
Immunostaining of Chimeric Organoids
2
Immunofluorescence Staining of Cells
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Coverslip cultures were washed with PBS, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.05% Triton X-100, and incubated with 3% bovine serum albumin (BSA) in 0.05% Triton X-100 for 2 h. The primary antibody was applied at 4°C overnight, followed by the secondary antibody for 2 h. Next, the coverslips were stained with 4% 6-diamidino-2-phenylindole (DAPI; 2.0 mg/mL) for 10 min at room temperature and mounted. Expression of BrdU and TFAM was determined using monoclonal or polyclonal antibodies conjugated to Cy3 and Alexa 488, and a goat-anti-mouse IgG secondary antibody conjugated to Alexa 555 or a goat-anti-rabbit IgG. The cells were visualized under a fluorescence microscope, and images were obtained using an AxioVision imager and analyzed in silico (Carl Zeiss MicroImaging GmbH, Jena, Germany).
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