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).
Ls 510 meta
Manufactured by Zeiss
Sourced in Germany
The LS 510 Meta is a laser scanning microscope system developed by Zeiss. It is designed for high-resolution imaging and analysis of samples. The system utilizes a confocal setup and features a multi-channel detection system.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Rabbit anti wt1, by Santa Cruz Biotechnology (1 mentions)
Species specific secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Axio vision imager, by Zeiss (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Axio observer z1 fluorescence microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Tritc conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Cy3 conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using ls 510 meta
1
Immunostaining of Chimeric Organoids
2
Immunofluorescence Staining Protocol
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Cells were fixed with phosphate-buffered saline (PBS) containing 4% paraformaldehyde (Società Italiana Chimici, Rome, Italy) for 15 min at room temperature (RT). After three washes with PBS, the cells were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) for 10 min at RT (when necessary) and then incubated with 5% bovine serum albumin (BSA, Sigma-Aldrich) for 1 h as a blocking solution. The samples were stained with primary antibodies diluted in 2% BSA solution overnight at 4 °C, followed by the corresponding Alexa 546- or Alexa 488-conjugated secondary antibodies for 1 h at RT. F-actin filaments were stained with TRITC-conjugated phalloidin (Invitrogen). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Images were taken using an inverted confocal laser-scanning microscope (LS 510 Meta) or the Axio Observer Z1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The primary antibodies used are listed in Table 2 .
3
3D Confocal Microscopy of Microtubules
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Digital z-stack images of WGA-lectin-labeled microtubules were acquired with the LS 510 Meta inverted confocal laser scanning microscope (Carl Zeiss) and were reconstructed into a 3D image through AxioVision 4.8 imaging software (Carl Zeiss).
4
Quantification of Sirt3 Protein Expression
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Paraformaldehyde-fixed cells were incubated with Triton 0.3% and, after blocking, with a rabbit monoclonal anti-Sirt3 antibody (1:100, Cell Signaling Technology Inc., Danvers, MA), followed by a goat anti-rabbit Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Baltimore Pike, PA). Nuclei were stained with DAPI (Sigma-Aldrich). Sirt3 staining was examined under confocal laser scanning microscopy (LS 510 Meta, Zeiss). Sirt3 protein expression was quantified in 10 randomly acquired fields per sample. Specifically, the area corresponding to the Sirt3 staining was measured in pixels2 using Image J 1.40g software and normalized for the number of DAPI-positive nuclei.
5
Immunofluorescence Labeling of Mouse Soleus Muscle
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Soleus muscles from 6-month-old C57BL/6 mice were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 2 h at room temperature. Small bundles were washed twice with PBS and blocked for 1 h in PBS containing 1% BSA, 10% goat serum and 0.5% Triton X-100 (added to permeabilise the membrane). They were incubated overnight at 4°C in primary antibody and the following morning they were washed 3 times in PBS. They were incubated with the secondary antibody for 1 h at room temperature before being mounted on coverslips with anti-bleach media (Slowfade Gold antifade reagent; Invitrogen (Molecular Probes) Eugene, Oregon, USA). Primary antibodies were: anti-RYR1 (Ryanodine receptor type 1), 34C (dilution 1:30; Developmental Studies Hybridoma bank, University of Iowa); rabbit monoclonal anti-GR (dilution 1:100; Abcam). Secondary antibodies were: Cy3-labelled goat anti-rabbit IgG (dilution 1:300) for single GR labelling; Cy5-labelled goat anti-mouse IgG (dilution 1:200) and Cy3-labelled goat anti-rabbit IgG for double labelling (dilution 1:300). All secondary antibodies were from Jackson ImmunoResearch Laboratories, Lexington, KY. Specimens were finally viewed using a scanning laser confocal microscope (LS510 META; Carl Zeiss, Jena, Germany).
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