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Comparative Physiology @ Guelph

EM Protocol for Fixing and Embedding Cells on Silastic Strips

  • Refer to standard fixation protocol for TEM for instruction on making required stock solutions.
  • Make 1% uranyl acetate solution (0.1g in 10mL) and various EtOH solutions for the series
  • Refer to live cell stretching protocol for instruction on cell mounting procedure, using two strips per stretching trial and generous amounts of stopcock grease (follow until step 23, when grease is added)
  • Calculate final strain and stretch strip to desired length
  • Fix for 2 hours in 1.5% para/1.5% glut in cacodylate pH 7.3 at room temp
  • Wash 3x with 0.1 M Na cacodylate pH 7.3 (10 min each wash)
  • Post fix in 1% OsO4 in 0.1 Na cacodylate for 2 hours at room temp
  • Wash 3x w/ dH2O (10 min each wash)
  • Stain 2 hours with 1% uranyl acetate solution in the dark, at room temp
  • Wash 3x with dH2O (10 min each wash)
  • Dehydrate with EtOH series

30% EtOH 10 minutes

50% EtOH 10 minutes

70% EtOH 10 minutes

95% EtOH 10 minutes

3x100% EtOH 10 minutes

  • Infiltrate with 1:1 Polybed:EtOH overnight
  • Replace 1:1 with three changes of neat Polybed (30 min each)
  • Place strip covered in neat Polybed into 60 degree oven for at least 24 h

 

Total time for procedure (not including making stock solutions): 8.5 hrs, 10 min

Last modified by Whitney Moore, August 2007.