10:30A — 11:15 P
- Overnight confocal quick check data look great.
- Day 3 in situs (using new chambers).
- WaP book6, ch23
- Labeling secondary antibodies. all are donkey antibodies from Jackson Immuno. reported are absorption values from the spec at the corresponding wavelength peaks.
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- rb 750. 750=.80, 405=.18,280=.447. 3.8:2.4, con 1.3 uM
- gt 647. 647=. 463, 405=.198,280=.419 3.9:1.3 1.3 uM
- shp 750. 750=.661,405=.175,280 =.429 3.8:2.0 1.3 uM
- gt 488 488 = .138, 280=.339
0.99 dye/antibody. - shp 647: 647=.450,405=.212,280=.460 4.4:1.3, 1.4 uM
- m 647: 647=.573,405=.216,280=.50, 4.0:1.5, 1.5 uM
- rb 488: 488 = .22 – .07 bkd. 280 = .448-.07 bkg.
0.96 dye/AB. 1.7 uM - m 488: 488 = .125, 280 = .25
1.2 dye/AB 1.3 uM - shp 488: 488 = .109, 280 = .354
1.6 uM 0.68 dye/AB = trash
- concentrations and ratios computed with Graham’s calculator
- Moved ‘Protocols’ to GoogleDocs
- 7:40p Confocal laser heating up. Found control should play around with it.
- No success engaging direct focus. MP08 snaD from 2-17 uses LacZ-dnp (poor choice). some embryos found anyway.
- System may have drifted back in Z during attempt at engaging focus drive and figuring out color channels. Dropped 488 for speed, doesn’t seem to image well in 4 channel setup.
- in situs stained and in final PBT rock at 4C O/N. Tomorrow check staining on Zeiss700 and post-fix good embryos — proceed for embedding.
- Found rat anti-DNP, free up rabbit channel for in situ / protein stain.
- Set up 2 new wt cages (11:00P).