10:20 A
Fly work
- New cage of MTD (not crossed, just as control). Use for Pc and Su(Z)12 staining. Methanol treat after primaries to improve in situ.
DNA FISH
- Day 3: DNA-FISH + antibody.
- [spreadsheet 0AjSqkxgziU1YdG90a29UaU5ua1FLUXZ5LTFmM1pMSlE sheet=8 602 302]
- postfix 1 hour with new post-fix solution
- confocal test stains. shp-H3 is not great. DNA-FISH dots are clearly present in centromere samples. Hopefully is stays through embedding this time.
- 405 from rb-750,405 to K27me3 looks blank. maybe 750 isn’t labeling well with 405 despite peak (it’s quite a small peak, easy to mis-estimate?). So far 750 STORM switching has been fine though.
- 405 from H3-750,405 looks great though.
- Not much dot evident in 3R fragment paint, probably reused and diluted probe too much.
Other
- Contact NIH director about U13 application for BIRS workshop
Data analysis
- Rewrite K27me3 cluster analysis as function findclusters, which requires overlap by with nuclei but does report results per nucleus. Suspect average cluster size and cluster density as a function of average within an nucleus and average of nuclear averages overweights clusters in small cells compared to large cells.
- see post
100 mL post fix solution (.1% GA, 4% PFA, .1% Tween, in 1x PBS)
- 100/.7 uL 70% GA = 143 uL of 70% GA
- 4/.1 mL PFA = 40 mL of 10% PFA
- 1 mL 10% Tween20
- 10 mL 10x PBS
- fill to 100 ddH2O
DNA