Sunday 12/09/12

10:20 A – 7:30P, 9:30-11:00P


  • 750 1600 mA = 18 mW
  • 1.45 mW 405
  • 200 mW set 647
  • Made new Glox
  • 750 not switching well.  Not sure if it’s just low labeling (405 looked okay but not strong).
  • 647 is high background.  Maybe too much out of focus non-specifically bound dye.  Should do higher temp hot wash.
  • changed to new aliquot of BME.  Seems to provide better contrast
  • 488 much better after ~10,000 frames of bleaching.  Better contrast.  Should manually analyze a few more of these to get better fits.

Summary Hp1a, centromere

  • not strongly colocalized in S2 cells, but Hp1a is very weak.
  • well colocalized in polytene cells but HP1a staining is still pretty weak and fuzzy
  • This weak Hp1a staining contrasts strongly with the excellent Hp1a staining tested on embryos 8 months ago.  Probably the antibody has started to go bad.  Should make glycerol frozen stock.  Fill out paperwork to order new HP1a
  • Also ordering to test: anti-BEAF and anti-GRO

Cells culture cell line dead.  Need new cells.  Maybe best to wait until after the break?

Stain then cryo-section: observations

  • Dm01 stain survives sectioning.  H3K9me2 didn’t really work in the first place but has some signal.
  • centromere signal surprisingly not detectable at all (was very strong by confocal)
  • 2 good adjacent sections near one corner of the coverslip.  No other obvious sections noted.  (Maybe spent too much time cutting blank gelatin?)

New Centromere staining

  • 11/27/12 fixed S2 cells
  • and previously labeled and stained MTD embryos from new cryo section.
  • both centromere labeling O/N



  • wrote 3D functionality (by color) into STORMrender.  (capable of rendering multiple channels in graded color.  Default map is hsv).


  • En germband extension embryo analysis



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