11:00A – 12:15 A
- in situs day 3
- STORM imaging: Invitrogen Po-Po family dye 5 and dye 6 do not seem to survive embedding process — see outline of nuclei nicely but no DNA staining.
- H3K27ac sections show punctate spots in nuclei rather than glowing nuclei.
- 10 / 15 min etching not always sufficient to remove all resin. Partially remaining resin fluoresces brightly under strong 633, causing sample to give nice conventional images and impossible to image for STORM. might be issue with H3K27ac slides as well.
- Discussions with Yari: antibodies don’t penetrate GA fixed tissue well. Keep for post-fix only?
- Try adding DNA-dyes to sample after etching.
- Fix embryos, 15 min, 8% formaldyhde: 2-3hr, 3-4hr, 4-5hr, and 5-6hr.
- flip collection plates on wt and MP08 cages.
- make new secondary antibodies, see antibody log. (not made yet).
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- 647 1.5uL -> 1.4-2 dyes/antibody
- 750 2uL -> 2 dyes/antibody
- Atto 488 4.5uL -> 2 dyes/antibody. 3.5 -> 1.2 dyes/antibody
- Mount in situs slides 4-6. others at 4C in PBS.