10:00 A -7:30P
STORM
- Finish O/N STORM run
- Recorded 9 200,000 frame movies in very close proximity regions. Last image no signal — focus issue or bleaching?
Probe making
- analyzing gel of test digest
- Ethanol precipitating and re-supending nicked probe
Sequential washes
- STORM image cy5 tile (need to analyze still)
- sequential image of Cy3 tile. Image with feducial marks – 715/755 beads
- washout cy3 tile 1 min, 50% formamide 65C. Image. Some background still, may not be specific signal. some 715/755 beads move/hop positions
- washout 15 min 50% formamide 65C. Possibly slight but non-dramatic improvement in reducing signal. some 715/755 beads move/hop positions
- restain 3 hrs
- reimage same cells. signal not restored. some cells have bright nuclei.
Conclusions
- 65C 50% formamide wash may be good for DNA, too harsh for RNA.
Next steps, wash out and restain
- label cells in 4 different chambered slides
- washout probe at 55C 50% formamide, 50C 50% formamide, 45C 50% formamide, 40C 50% formamide for 3hrs and O/N
- image washout.
- restain each sample for 3 hours. Test stain fresh sample for 3 hours.
- Re-stain O/N if necessary
- Take successful restained samples and repeat.