Uranyless cryo-acetone
new version stain on bloc for AFS, Cryo application. 
Protocol : UranyLess cryo-Acetone version cryo for AFS application stain sample on bloc:
Culture BeWo cells on Saphir disc cryofixed by EM HPM 100
picture of Uranyless acetone
- 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
picture of microscopy results uranyless acetone
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 acetone staining, membrane boundaries are not visible.
