8:45 A — 7:30 P, 8:45P – 12:40 A
- Finish O/N confocal run
- transfer files
- modify imviewer LSM to automatically detect all .lsm files in a folder and convert them all to TIF / labeled stacks of TIFs automatically.
- start code to analyze dot distances for Dfd-Tub data.
- Would be better with clear nuclear outlines. Should have stained nuclear lamina.
- Should also take overview images a 40x full field (or 20x?) for better staging.
- Upgrading Matlab to 2012b.
- water recombineering crosses (need new syringe + ddH2O).
- set up new cage of MTDs
- MTD x Su(Z)12s F1s so far no viable eggs. Should collect F1s and fix some embryos
Cryosection staining / DNA-FISH
- staining worked well, sections still flat
- brief SSCT wash, brief PBT wash, block 1hr.
- 50 min in primary antibody (rb H3K9me2 + m Dm01-Cy3B)
- 1 hr (not more) wash in PBT (minimize opportunity for sections to detach).
- incubating in secondary (Abcam anti-rb 750).
- check on Turnkey, no 405 label obvious.
- Fails 750 on STORM2 (see below).
- Restaining O/N rb-488. (how could the abcam 750 fail? or maybe the dyes have died?)
Wholemount embryo staining / DNA-FISH
- DNA FISH O/N program finished, samples at RT.
- exchanged labeling solution for post hybe, take sample of embryo to check labeling on Turnkey. — labeling looks good.
- Started 37C rinse in post-hybe 2 (50% post-hybe 1 + 40% formamide in PBT) (~20 min)
- 20% formamide in PBT rinse also at 37C (~20 min).
- Rinsing at RT in PBT.
- Block, primary antibodies rb H3K9me2 + m-Dm01 (unlabeled) at RT 2+ hrs.
- Rinse RT
- secondaries: a-m cy3B, abcam anti-rb 750
- Embedded embryos in small volume of 12% gelatin.
- Incubating in 50% sucrose solution O/N (should have saved some embryos to check on confocal).
- pour PAGE denaturing gel to separate bands for probe.
- Remember to heat denature first this time!
- Still only single band. Maybe denaturing gel did work last time. Last time band was much tighter. 160 uL of probe with + 170 uL 2x loading buffer to nearly 400 uL sample needed two wells. not as bright and a little more spread out.
- Gel crushed and DNA redissolving at 55C in 2 tubes per band O/N.
- Will precipitate and test tomorrow.
- Send Order probe library from LC to Matt for PO.
In situs day 1
- using YW O/N embryo stock
- inv/en-DNP, tou-bio, E(pc)-dig
- dfd-bio, tub-dig
- use directly labeled Dmo1 to mark nuclear lamina
- got poly-lysine from hazen (Wenqin, properly stored, never opened, newly recieved)
- high static light white plastic/styrophome like crystal.
- made .5 mg/mL (25 mg in 50 mL) stock solution in PBS pH 8.5 (probably should have used tris). coated coverslips — dry cloudy.
- centromere stain switching very well. Some sections well attached.
- No trace of 750 (or 405 on turnkey) at all
- staining O/N at 4C with a-rb 488. I don’t understand why this doesn’t work.
- O/N imaging of Dfd/tub
- took overview of full field 40x (70%-whole embryo) for staging. Imaging 3 channels at speed 6, 22 positions, 35 heights. Hopefully should be done by 10A.