Cells grown on glass cover slips were fixed with 4% paraformaldehyde in PBS for 15 min. After washing in PBS, the cells were permeabilized in 70% ethanol overnight at 4°C. They were then re-hydrated twice for 5 min in 2× SSC containing 10% formamide. The precursors to the 18S rRNA were localized by fluorescence in situ hybridization (FISH) with a 5′ ITS1 probe coupled to Cy3 (35 (link)). The cover slips were washed in 2× SSC containing 10% formamide, and DNA was counter stained with Hoechst 33342 (Invitrogen), before mounting in Mowiol 4.88 (PolyScience, Inc.). The slides were observed with an inverted microscope (IX-81; Olympus), equipped with a ×100, oil immersion objective (Plan Apochromat, 1.4 NA; Olympus) and an Orca Flash 4.0 camera with a pixel size of 6.5 μm (Hamamatsu), driven by MetaMorph (MDS Analytical Technologies).
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Fluorescence In Situ Hybridization of 18S rRNA
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Corresponding Organization : Université Toulouse III - Paul Sabatier
Other organizations : ETH Zurich
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Variable analysis
independent variables
- Fixation method (4% paraformaldehyde in PBS for 15 min)
- Permeabilization method (70% ethanol overnight at 4°C)
- Re-hydration method (2x SSC containing 10% formamide)
- Localization of 18S rRNA precursors by fluorescence in situ hybridization (FISH) with a 5' ITS1 probe coupled to Cy3
- Cell growth on glass cover slips
- Washing in PBS
- DNA counter staining with Hoechst 33342
- Mounting in Mowiol 4.88
- Microscope equipment (inverted microscope IX-81, 100x oil immersion objective, Orca Flash 4.0 camera, MetaMorph software)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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