11:00 A – 7:30 P, 9:00 P – 11:00 P
Trouble-shooting hybes
- check samples:
- AATAT-P1 + S1-short on cells – strong and clear
- en-P1 + S1-short on cells – clear staining, bleaches fast
- en-RNA-P1rc + S1-short on cells – no staining at all
- embryo sections (previously failed BX-C stain) en-P1 maybe stains?
- embryos sections 2 (previously failed BX-C stain) AATAT-P1, no staining evident. maybe sample too degraded (cut in Jan and hybed for the first time back in feb. In 2x SSCT at 4C since. AATAT stained controls from that time still look reasonable).
New cell staining
- Finish prepping cells for hybe. (cell density unfortuantely quite low).
- Moved cells to 20% glycerol, snap-froze in LN2 2x, wash, HCl treat, wash in SSC, now in prehybe.
- samples listed below
- denatured on downstairs heat block set to 88C (approx temp ~85C)
| Sample | primary | secondary |
|---|---|---|
| RNA hybe mix | 5 uL en-RNA-P1rc in 25uL | 1.5 ul S1* |
| high conc | 20 uL en-RNA-P1rc straight | 1.5 ul S1* |
| low conc | 1 uL en-RNA-P1rc in 29uL | 1.5 ul S1* |
| Yellow | 5 uL in 25 | 1.5 ul S1* |
| Black | 5 uL in 25 | 1.5 ul S1* |
| Green | 5 uL in 25 | 1.5 ul S1* |
| Green high | 5 uL in 25 | 1.5 ul S1* |
| Ubx | 5 uL in 25 | 1.5 ul S1* |
*2nd batch, reconcentrated short secondary
Cell cryo?
- Goal: get nuclei closer to coverglass to reduce media mismatch distortion and improve SNR.
- pellet cells at 1300 rpm (~200g) for 5 min
- Remove medium. Resuspend in 5% formaldhyde in .5x PBT for 10 min. Spin down
- rinse in PBS
- spin down
- rinse in PBS
- spin down
- move to 12% glycerol, incubate 1 hr
- move to 4C to harden glycerol. Cut out of 1.7 mL eppendorf, move to 30% sucrose
- alternative method for cell-ultra-cryo to try next time if this doesn’t give good results. Basic idea to is cover (fixed) cells with 12% gelatin and cut out and mount these blocks.
embryo staining
- day 2, washout en-primary antibody
- re-block 1 hr
- antibody incubation in dark, m-488-cy3b
STORM analysis
- dual color Ph analysis finished running
- set up bead analysis to compute chromewarp file.
- split-dax chromewarp approach is defintely slower, takes hours to run. Should see if we split first and then call Rundotfinder on all movies of a common color channel, if it goes faster. Should also figure out how to load multiple dax in parallel. Current process is using 10-20% of CPU only.
- set up analysis of S4 and S5 hybe condition tests on S2R+ cells.
Other
- select some images for H3K27me3 density dynamics for SDB
- update letter format version of BJ review and send to Jeff