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Uranyless acetone   

new version stain on bloc for AFS, Cryo  application. Formvar solution

Protocol : UranyLess Acetone version cryo for AFS application stain sample on bloc:

Culture BeWo cells on Saphir disc cryofixed by EM HPM 100

  • 8 hours 2% OsO4/acetone solution at -90 °C
  • 0.5-hour temperature increase
  • 8 hours 2% OsO4 /acetone solution at -60 °C
  • 0.5-hour temperature increase
  • 8 hours 2% OsO4 /acetone solution at -30 °C
  • Remove OsO4 solution and add pure acetone
  • 1 hour washing
  • Remove acetone and add UranyLess acetone version cryo for AFS application.
  • 8 hours UranyLess acetone version at -30 °C (stain on bloc)
  • 1.5 hours temperature increase
  • 0 °C End of AFS

As soon as 0 °C was reached, the samples were washed one time with acetone glass distilled and subsequently embedded in Epon resin at room temperature.

this protocol developed by Delta Microscopies R&D and validated in the laboratory of the Faculty of Medicine of Graz Austria

 

 

     

OsO4 fixation without uranyless cryo staining, membrane boundaries are not visible.