Achieve excellent freeze substitution results in as little as 90 minutes with this unique kit.
- Heater block for 12mm tubes (13mm block is also available)
- Ice chest
- Temperature probe
Components can also be ordered separately.
Why does it work?
Freeze substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. K.L. McDonald and R.I. Webb introduced a simple and effective method for freeze substitution1 .
- In well-frozen samples water molecules do not move around very much, even as the temperatures rise to a point where hexagonal ice formation is expected.
- Agitation speeds up the substitution of acetone for water molecules .
- Water in the substitution mixture does not appear to slow down substitution, but helps improve membrane contrast
Does it work for all samples?
- If a sample can be successfully freeze substituted by the old methods, then the quick FS method should work just as well.
- McDonald and Webb have used this procedure with complete success for over a year and a half for all the samples they have freeze substituted.
- If samples show evidence of ice damage then it is because they were damaged during freezing and not during freeze substitution.
Warming Curve With and Without Use of Lid
Typical temperature curves using the EMS Freeze Substitution Kit. With the lid OFF the time to 0°C is about 2 hours. With the lid ON the time is about 3 hours. Results may vary depending on the particular setting of the shaker. For example, hood air flow can have a definite influence on the shape of the curves.
- The equipment should be used in a fume hood in case there is a leak of osmium-acetone during a run. We suggest doing a trial run with acetone only in the cryovials to make sure that they are sealed correctly.
- When sealing cryotubes that contain frozen fixative and sample, use a warm cap so that the O-ring is flexible and gives a good seal.
- Take care when removing the caps after a FS run because there is some pressure built up inside the cryotubes and you can spray osmium/acetone on your hands if you are not careful. Cover the cap with a piece of lab tissue when removing and wear gloves.
Cross-section through nerve cells in the head of C.elegans.
A BHK cell processed in 90 minutes by the quick FS method. Abbreviations: CCP = clathrin-coated pit, Cav = caveolae, EE = early endosome, IF = intermediate filaments, LE = late endosome, M = mitochondrion, MT = microtubule. Bar = 1 µm. Modified from McDonald and Webb (2011).
- K.L. McDonald* and R.I. Webb**, *Electron Microsocpe Laboratory, University of California , Berkley, CA and ** Centre for Microscopy an Microanalysis, University of Queensland, Queensland, Australia