Cryofixation uses liquid nitrogen to create ultra-rapid freezing, fully fixing a sample in milliseconds, and mainitaining internal structure. Following freezing samples are embedded in resin, normal embedding processes are not appropriate due to the fixation method, therefore a protocol called freeze substitution is used. Once embeded samples are proccessed as normal.
A method used to replace all the water contained in a sample with acetone. Done by submerging samples in an acetone solution and slowly warming the specimens from -196 degrees celsius to room temperature. This water is removed to stop damage being caused by the fomation of ice crystals, which form as the temperature is increased. After freeze substituion normal embedding methods can be used.
The method outlined was used for the preparation of C. Elegans;
– High Pressure Freeze Sample, in our lab we use a Leica EM HPM100 to freeze our samples.
– Freeze Substiution
> Immerse samples in freeze substitiuion solution of osmium and dry acetone, this is done under liquid nitrogen at -196 degrees celsius.
> Remove liquid nitrogen and replace with dry ice. Place on a shacking rack and leave overnight.
> Remove dry ice and allow the sample to warm up to room temperature.
– Infiltration with resin (Epon)
– Polymerisation at 65 degrees C overnight
Cross section through C. Elegans, embedded using high pressure freezing
and freeze substituion.