Wednesday 02/22/12

9:30 A – 8:50 PM

  • Sectioning: 300 nm. trapazoids work better, long edge at bottom.
  • Cut 3 coverslips of S6.  Two at 330 nm, one at 100 nm.
  • Coverslip 1 at 330 nm is tiled from upper left to lower right in continuous sections (~100+ sections).
  • coverslip 2 has several sections of 70 nm at bottom.
  • Confocal of S6 — signal relatively weak by confocal.
  • Confocal of 100 nm sections of S1 (hox + H3).  561 and 405 invisible by ocular even on 63x.  All channels very weak for confocal scan, lasers up full.  Much weaker than 300 nm section.
  • Identified transcribing nuclei in slide S6 (hox + Pc).  Circled.  Take STORM image of this embryo.
  • Fix embryos: MP09 (1.5-5.5 hrs), sna 4x (1.5 – 5.5 hrs), and wt (3.5-7 hr).  wt and MP09 were 10 min fix.  sna 4x is full 25 min.
  • Attempt STORM analysis of 3D movie.  Need calibration file.  Need to make calibration file from bead slides, then run insight analysis with elliptical particle finding, and then run Sung-He’s Matlab script to create a calibration file which can be uploaded in Insight.
  • Use short-fix wt for staining Pc (2nd round with pre-diluted PcG antibodies) and short fix MP09 bcd and bcd + hb at 4C O/N
  • 9PM rad21, H3 shp/rab and H3-4color samples at 70C.
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