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.