Fixation of GFP-tagged proteins
Materials
- Fixing Solution (4% paraformaldehyde, 3.4% sucrose)
- Wash Solution (100 mM potassium phosphate pH 7.5, 1.2 M sorbitol)
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Method
- Spin down 1 ml of cells at a density of 5⋅106 cells/ml in a 1.5 ml tube and remove the supernatant
- Resuspend in 500 µl of Fixing Solution and vortex
- Incubate at room temperature for 15’
- Spin down cells and remove supernatant
- Wash once with 1 ml of Wash Solution
- Resuspend in 250 µl of Wash Solution At this point it is possible to store the cells at 4ºC for 2-3 weeks
- Briefly sonicate cells just before the observation (~1-2 min in waterbath);