Sunday 11/18/11

10:40 A -8:40 P

  • Troubleshooting sodium ethoxide etching:
  • 2 washes of 30 min each gives good etching, 100 nanometer sections of embryos are still visible, but resin is gone.  Resin otherwise still visible by confocal.  not readily detected on STORM2.
  • Testing post-etching of DNA dyes.  Broke one coverslip, etched new coverslip.
  • Invitrogen DNA dye-540 does not strongly label DNA in sections
  • test dye in whole-mount embryos, strongly labels everything except nuclei  🙁
  • Hoechst doesn’t seem to label too well: either DNA too dried out or embryo background in 405 after GA post-fix and embedding is too high.
  • No sign of RNA staining in MP09 y(i) and y(full) labeled embryos.
  • Checking alignment of pan-histone stained super-resolution imaged nuclei, some sections which are too thin on left side are also compressed along the left-right axis.
  • engrailed and hox stained embryos: Rinse out post fix, dehydrate into ethanol and start resin embedding.  in 50:50 resin:EtOH O/N.
  • washing out secondary labeled bcd, ftz, and K4 embryos.  check labeling on Zeiss700, DNA dyes still look good, can’t see hb (labeled in 750, not showing up in 405 but has to compete with 647:405 and not sure if this 405 can fluoresce much anyway in conjugate).
  • engrailed-555 not readily detected in ftz-labeled embryos.  No idea why.
  • WGA-cy3B (thanks Yari) to bcd, WGA 750 to ftz, and WGA 647,750 to bcd, ftz. Label reaction for 1hr.  Start rinses, more rinsing in PBT at 4C O/N.
This entry was posted in Summaries and tagged . Bookmark the permalink.