The cellular uptake of XOs and P-XOs into cells was visualized with a LSM710 confocal laser scanning microscope. Additionally, cells were tagged with fluorescent antibodies. In brief, four-well plates were fixed with 4% paraformaldehyde for 20 min. Protein was blocked with blocking buffer containing 0.1% saponin (DAKO; Agilent) for 1 h at room temperature. Then, the cells were incubated with the following primary antibodies diluted in blocking buffer at 4 °C overnight: VE-Cadherin (ab7047), vWF (ab6994), and Ki67 (ab15580). After three PBS washes, the cells were tagged with fluorescent secondary antibodies for 1 h at room temperature. Then, slides were mounted with Prolong Gold mounting solution with DAPI, and imaged with an LSM710 confocal microscope.
Saponin
Manufactured by Agilent Technologies
Saponin is a naturally occurring compound extracted from various plant sources. It functions as a surfactant, reducing the surface tension of liquids and facilitating the formation of stable emulsions. Saponin is commonly used in laboratory applications to aid in the solubilization and dispersion of other compounds.
Automatically generated - may contain errors
Lab products found in correlation
Saponin, by Merck Group (2 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Airyscan detector, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Goat serum, by Agilent Technologies (1 mentions)
Anti sag1, by Abcam (1 mentions)
Cy3 conjugated goat anti mouse igg plus igm, by Jackson ImmunoResearch (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Goat anti mouse fitc, by Agilent Technologies (1 mentions)
Swine anti rabbit fitc, by Agilent Technologies (1 mentions)
3 protocols using saponin
1
Visualizing Cellular Uptake of XOs and P-XOs
2
Immunofluorescence Imaging of T. gondii
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Cells were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) permeabilized with 0.02% saponin (Calbiochem-Merck) and blocked in 0.002% saponin with 2% goat serum (DaKoCytomation). The outer membrane of T. gondii was visualized by anti-SAG1 (Abcam) at a concentration of 1/700. As secondary reagents, 1/200 concentrated Cy3-conjugated goat anti-mouse IgG plus IgM (Jackson ImmunoResearch Laboratories) or Alexa Fluor 633-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) was used. Nuclei were counterstained with 1/2,500 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). The coverslips were fixed in fluorescence mounting medium (Fluoromount-G; Southern Biotechnology Associates). Fluorescence was visualized using an LSM780 confocal microscope with Airyscan detector (Zeiss) using a 63× Plan-Apochromat oil immersion objective (numeric aperture, 1.4). Image analysis and processing were performed with ZEN (Zeiss) and Imaris (Bitplane).
3
Microvesicle Isolation and Labeling for Shiga Toxin
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HeLa cells were stimulated with Stx1B:Alexa488 or Stx2. Microvesicles were isolated and labeled with mouse anti-CD44PE and rabbit anti-Stx2 (BEI resources, Manassas, VA) and swine anti-rabbit FITC (Dako, Glostrup, Denmark), both diluted in 0.1% saponin (Sigma-Aldrich). Platelets were stimulated with Stx1 or Stx2. Microvesicles were isolated and labeled with mouse anti-CD42PE and mouse anti-Stx1 (Santa Cruz Biotechnology, Dallas, TX) and goat anti-mouse FITC (Dako), both diluted in 0.1% saponin, or rabbit anti-Stx2 as above. The procedure is detailed in the supplement.
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