The protocol for adherent mammalian cell lines was optimized for Stellaris FISH probes. Hybridization was performed overnight and no anti-fade was used for imaging. The sequence of the CAL Fluor Red 590 tagged probes targeting the CYP19A1 mRNA can be provided upon request. Samples were imaged using a Nikon Ti-E scanning laser confocal inverted microscope (A1) with 60x oil objective in tandem with Nikon NIS-Elements imaging software. Excitation was by 561.5 nm diode-pumped solid state. Detection was via 595-50 nm filter. Optical sections were captured at 0.300 μm intervals and a resolution of 256 by 256 pixels and zoom factor of 6.8, resulting in a voxel size of 0.0047 μm3 (0.1243 μm by 0.1243 μm by 0.3 μm). Four times averaging was used to reduce photon and camera noise. An automated spot count algorithm determined the number of mRNA 32 (link). For the analysis we included 30 positive control cells to better define an mRNA spot.
Protocol detail
Optimized Adherent Cell Imaging Protocol for Stellaris FISH
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Cell lines
Cells
Cyp19a1
Factor 8
Fish
Hybridization
Mammalian
Mrna
Scanning laser confocal microscope
Sections
Corresponding Organization : University of Milan
Other organizations : European Institute of Oncology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, University of Amsterdam, Lawrence Berkeley National Laboratory, University of Calabria, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg Institute for Stem Cell Technology and Experimental Medicine
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Variable analysis
independent variables
- Hybridization duration (overnight)
- Number of mRNA spots
- Usage of Stellaris FISH probes
- Lack of anti-fade for imaging
- Microscope (Nikon Ti-E scanning laser confocal inverted microscope with 60x oil objective)
- Imaging software (Nikon NIS-Elements)
- Excitation wavelength (561.5 nm diode-pumped solid state)
- Emission filter (595-50 nm)
- Optical section interval (0.300 μm)
- Image resolution (256 by 256 pixels)
- Zoom factor (6.8)
- Averaging (4 times)
- 30 positive control cells
- Independent variables not explicitly mentioned, except for the hybridization duration.
- Dependent variables not explicitly mentioned, except for the number of mRNA spots.
- Control variables not explicitly mentioned, except for the items listed above.
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