9:10 A — 8:00P, 10:30P – 12:45 A
- collect prepped samples
- 10A – 2P, cutting ultra cryo sections of 1-10hr MTD DFISH-fixed embryos
- For next time:
- denser embryo packing in smaller gelatin sections
- Cryo protect at least 30 min in 2.3 M sucrose
- bring Diamond Cryo Knives (in Hazen’s drawer downstairs)
- Try 22 mm coated coverslips. These fit into the 6-well dishes to make washing easier.
- storage — cut slits in a styrophome, imbed edge of coverslip
Staining ultra cryo sections
- Coverslip 1-2: sections dehydrating at 42C on hotplate. (4P – 10P. sucrose outline still present at 10:30, but samples visible and seem to be adjacent to glass, let’s hope they stick).
- Coverslips 1-2 incubating in PBT, 10 min.
- Incubating in 2x SSC 10 min.
- Incubating in RT 2X SSC + 50% formamide, 10 min. (should confirm if this current batch is 4X SSCT + 50 Formamide for 2X final…)
- Heat denaturing 3 min at 92 (probe 3 min at 95). Coverslips directly on heat block
- Add probe, denature a further 2 min.
- Coverslips back in box with extra (95C heated) probe on top. Sample in 37C room O/N.
- Coverslip 3: section straight to PBS, may have lost some sections. some good sections by Hoechst. Now O/N in primary antibodies: rb-Pc and m-Dm01 at 4C.
- check crosses sna x TM2/TM6 –> flip to new tubes . Emerge in >4 days.
- check crosses Pr/TM3,Hbz x Sp/CyO,Hbz –> flip to new tubes. Emerge in 2 days?
Cell Labeling, DFISH
- Hot washes
- cold washes
- antibodies O/N. As in:
- [spreadsheet 0AjSqkxgziU1YdG90a29UaU5ua1FLUXZ5LTFmM1pMSlE 602 302 sheet=9]
- G-wells: original rb 488 stain (against rb H3K9me2) is completely gone after post hybe. rb 488 completely failed to label in post hybe incubation. But 405 from rb-750,405 is glaring bright. (How can this be? it’s the only 405 on the sample. maybe it’s a background cellular structure glowing in 405 but does not appear to be so, seems nuclear. Will compare to H3K27me3 controls)
- riddle fixed: the Jackson rb backround dots are glaring strong in the 750. The Jackson rabbit is clean. Primary antibodies were not crosslinked by post-fixation prior to hybe treatment and were lost in DFISH hybe. (H3K27me3 looks just like the H3K9me2).
- Attempting to restain primaries in G-wells with H3K27me3 and H3K9me2. Will see tomorrow on B wells if primaries still work after DFISH treatment.
Cell labeling, antibody restain
- rinse out secondaries
- check staining — restain does not work. Maybe all sites occupied and just labeling of R&D antibodies is bad?
Probe making — Gel electrophoresis extraction
- Bryan’s protocol
- cast a 100 mL, 16% denaturing gel (see protocol above)
- Loading buffer: 96% formamide, 4% .5M EDTA
- 300 uL 10% frozen stock ammonium persulfate
- 150 uL TMED (in 4C under John’s bench)
- samples dissolving in H2O at 37C O/N