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