For staining, organoids were cultured in 8-well glass-bottom dishes (Ibidi). Organoids were washed in PBS for 5 min and fixed in 5% NBF in 0.1% Triton X-100 in PBS for 15 min at room temperature. Cells were washed with 0.2% Triton X-100 in PBS for 30 min and incubated in FLASH blocking buffer for 1 hr. Primary and secondary antibody incubations were extended overnight at 4°C with 1 hr washes in PBS after each incubation. For imaging, wells were immersed in fluorescence mounting medium (Dako). Images were taken on a Zeiss LSM 780 confocal microscope as described above (3D imaging).
8 well glass bottom dishes
Manufactured by Ibidi
The 8-well glass-bottom dishes are a laboratory equipment designed for cell culture applications. They feature a transparent glass surface at the bottom of each well, allowing for high-quality microscopic observation of cells. The dishes provide a reliable and consistent platform for various cell-based experiments and analyses.
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2 protocols using 8 well glass bottom dishes
1
Organoid Immunofluorescence Staining Protocol
2
Culturing Mouse Embryonic Stem Cells
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Wildtype (IB10, subclone of E14 129/Ola (Hooper et al., 1987)) and Rad54-/mouse ES cells (Essers et al., 1997) (link) were cultured on gelatinized plates (0.1% porcine gelatin (Sigma)). The culture media consisted of 50% DMEM (High-Glucose, Ultraglutamine, Lonza), 40% BRL conditioned medium, 10 % FCS supplemented with non-essential amino acids, 0.1 mM β-mercaptoethanol, pen/strap and 1,000 U/ml leukemia inhibitory factor (mouse).
For imaging, cells are seeded in 8-well glass-bottom dishes (Ibidi) which were coated with 25 µg/ml laminin (Roche) for at least one hour. About 30 000 cells in 300 µl medium were plated per well the day before the experiment. Cells treated with IR were irradiated in an Xstrahl RS320 X-Ray generator (195.0 kV and 10.0 mA) at the indicated dose.
For imaging, cells are seeded in 8-well glass-bottom dishes (Ibidi) which were coated with 25 µg/ml laminin (Roche) for at least one hour. About 30 000 cells in 300 µl medium were plated per well the day before the experiment. Cells treated with IR were irradiated in an Xstrahl RS320 X-Ray generator (195.0 kV and 10.0 mA) at the indicated dose.
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