9:45 A – 11:30 P
Confocal
- Test protein stain (didn’t look good in conventional images)
- H3K27me3 weakly detectable.
- H3K9me2 not stained.
- Psc, SuZ(12), not well stained.
- compared to DFISH S2 cell (slide #1 in record, 10/23) All channels (except 1:100 centromere?) are terrible. H3K27me3 doesn’t come close to previous quality staining. According to my notes, these were also 1/2x PBT 5% form fixed, so it’s not likely the fixation. (maybe a brief restoritive depoly of the form buffer at 60C would help). Maybe cells less happy? cells certainly much more dense.
- maybe R&D antibody quality is worse than Jackson anti-rabbit? Recent 750 and 488 have been R&D, 10/23 stain might have been previous Jackson anti-rabbit (S2 cells maybe don’t have the random dot problem that previous lot of jackson antibodies had. Original round of jackson antibodies didn’t do this either, maybe should try some more. A good anti-rabbit is absolutely critical).
- Bingo. Jackson 488 looks great. Attempting restaining previously labeled cells, staining is not comporable. postfix may have masked antigens?
STORM2 notes
Talk to Graham about STORM2:
- STORM2 only use the last 3 frames of 10 frame bead file. — first .7 seconds the z focus lock hasn’t settled down.
- aligning back optics (check?)
- getting good flat fields of beads on STORM2
- parameters files
- calendar
- 750 switching in old pho sample looks great. Didn’t complete etching (~20min, should have done 45?) 647 switches off but enough background resin).
Prep for UltraCryo tomorrow
- make 6% gelatin solution. Not very stiff. upgraded to 12% gelatin.
- hydrating embryos. Embed in embryos
- clean and coat coverslips with Lysine: Add 300 uL of lysine solution to face side of coverslips arrayed in culture hood on sterilized styrophome supports. Incubate for 30 min with UV light on. Then aspirate extra media back into stock (can be reused for more coating). coverslips back in cermaic holder. Air dry and finish setting overnight in glass container. move to transport container tomorrow to take to ultra EM core.
- Making 70% sucrose solution for cryo protection of gelatin embedded embryos
- 2 gelatin sections incubated 1 hr in 30% sucrose and O/N in 70% sucrose for cryoprotection.
Cell labeling
- hot washes
- cold washes
- labeling intact. Except signal by confocal doesn’t look very good. 🙁
- Fix new cells, 15 min in 5% FA in PBT.
- reduce background with sodium borohydrate treatment (1%), 15 min.
- short block (~25 min), primary antibodies (45 min), washes (1 hr), secondaries.
- staining looks good! yay!
Cell staining for tomorrow
- new 8-well just added cent probe for DFISH
- test 8-well, 2 K27me3, 2K9me2, 2 Pc, labeled with 488. Now in cent probe for DFISH
- DFISH first now post-labeled with a-rb 750 and a-rb 488 (both jackson), at 4C O/N