9:30A – 5:00PM
Embryo fixation
- 9:30A Flip collection plate
- Making new buffer A fix mix 10 mL, fresh. (Should have made 100 mL and froze aliquots?)
- 10:30A Fix embryos. Now in -20C in MeOH.
- Write to lab and Francis lab about starting S2 cell culture.
Staining
- remove Pc antibody/embryos from 4C for 1hr RT incubation.
- remove and store Pc antibody for reuse.
- Rinse out antibody (1hr+)
- post-fix (heat-reactivate post-fix mix at 60C), 5% form 1/2x PBT, 25 min.
- rinse out post fix.
- Dehydrate into MeOH and store at -20C
Data analysis
- computing per cell cluster properties (more data points than per-image).
- As suspected, not a very robust measure. sharp segmentation of cells is difficult, and more numerous less robustless estimated small cells (nuclear fragments) dominate the analysis.
- Basic trends still captured by analysis though.
- Organizing results in ppt presentation for Francis group meeting