Sunday 10/14/12

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.
  • 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.
  • 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



This entry was posted in Summaries and tagged , . Bookmark the permalink.