Wednesday 11/22/12

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.

Fly work

  • 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 
  • Reagents:
  • 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


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