To prepare sample slides containing fixed insect and mammalian tissue culture cells for FISH, 100 µl of a 1×105–1×106 cells ml−1 suspension in rich media was spotted onto a poly-L-lysine coated slide and allowed to adhere for 1–3 hours in tissue culture conditions (e.g. 37°C, 5% CO2 for mammalian cells). Slides were then washed in 1X PBS, fixed in 1X PBS + 4% (w/v) paraformaldehyde for 10 minutes, rinsed in 1X PBS, washed in 2X SSCT, washed in 2X SSCT + 50% formamide (v/v), and finally transferred to 2X SSCT + 50% formamide for storage at 4°C until use. For a stepwise protocol, please see the Oligopaints website (http://genetics.med.harvard.edu/oligopaints ); also see references 7 and 10. For STORM imaging, samples were prepared in the same way except that 22×30 mm #1.5 coverslips were used in place of microscope slides. For DNA-PAINT imaging, samples were prepared in the same way except that Lab-Tek II 8 chamber coverglass vessels (Nunc) were used in place of microscope slides and no poly-L-lysine was used.
Lab tek 2 8 chamber coverglass vessels
Manufactured by Thermo Fisher Scientific
The Lab-Tek II 8 chamber coverglass vessels are a laboratory equipment product designed for cell culture applications. They provide a convenient platform for conducting experiments and observations on living cells. The product features a borosilicate glass chamber with an 8-well configuration, allowing for multiple samples to be studied simultaneously.
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2 protocols using lab tek 2 8 chamber coverglass vessels
1
Preparation of Fixed Cells for FISH
2
Preparing Cell Samples for FISH, STORM, and DNA-PAINT
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To prepare sample slides containing fixed insect and mammalian tissue culture cells for FISH, 100 μl of a 1 × 105–1 × 106 cells ml−1 suspension in rich media was spotted onto a poly-L -lysine coated slide and allowed to adhere for 1–3 h in tissue culture conditions (for example, 37 °C, 5% CO2 for mammalian cells). Slides were then washed in 1 × PBS, fixed in 1 × PBS+4% (w/v) paraformaldehyde for 10 min, rinsed in 1 × PBS, washed in 2 × saline-sodium citrate (SSCT), washed in 2 × SSCT+50% formamide (v/v) and finally transferred to 2 × SSCT+50% formamide for storage at 4 °C until use. For a stepwise protocol, please see the Oligopaints website ( http://genetics.med.harvard.edu/oligopaints ); also see refs 7 (link), 10 (link)). For STORM imaging, samples were prepared in the same way except that 22 × 30 mm #1.5 coverslips were used in place of microscope slides. For DNA-PAINT imaging, samples were prepared in the same way except that Lab-Tek II 8 chamber coverglass vessels (Nunc) were used in place of microscope slides and no poly-L -lysine was used.
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