Friday, 12/07/12

9:45 A – 8:20 P

  • Stop O/N confocal
  • Prep for ultra sectioning
Discussion with W. Bender
  • Graded AbdB expression probably produced by combinatoral enhancer action (multiple enhancers leading to the stronger pattern)
  • early enhancers are somewhat segment like.  
  • Not known whether the same individual enhancers activate AbdB and AbdA.
  • Not known how early enhancers talk to PREs or maintenance elements — influence later 
  • Some ‘boundary element deletions’ (like the classic Fab7 deletion) also remove the PRE, thus the expansion of active expression may have more to do with the loss of the iab7 PRE.  (instead of the ‘strong iab-7 late enhancer being primed by the iab6 initiatior).  More recent experiments suggest a mixing of fates.  
Ultra Sectioning
  • Sections very flaky, higher temperature, thick sections fracture much less.  (remember butter comparison).  Sections harder to pick up.
  • *Very important*:  Remember to let sucrose loop cool substantially prior to attempting to pick up sections.  This prevents the sucrose droplet from getting stuck to the knife (with the target section) and enables better section pick up.
  • Metal trimming tool cutting way, way better at both 1, 2 and 3 um thickness — long smooth, strong ribbons.  
  • Despite several attempts, did not get sections on polyL slides from trimming tool cutting to stay stuck after Toulene blue staining.  Perhaps the sections cut didn’t contain embryos?  (another difficulty of cryo — can’t see embryos easily in the block).   Perhaps left too long on heat block and they crack off with with overdried sections of Toulene?   
  • Recommendation from Colenso:  borrow the light source from the dissection rig (light source on ultra cryo rig is mindsplittingly horrible.  
  • coverslip with two spots has SCM embryos (maybe?  didn’t manage to get sections to checkout on Toulene blue).
  • Attempted cutting with glass knife.  Sections still not nearly as well behaved as on the metal knife.  
Cell labeling
  • Removed probe
  • hot washes
  • in PBT O/N
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