Re: that sinking feeling

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>Karen wrote:
>I have a graduate student who has done some physiology on a monkey brain.  He
>now wants to cut cryosections of this brain (electrode tracks have been marked
>with DiI), and has put a portion of the brain (maybe a 3-4" cube) in 30%
>sucrose and is waiting patiently for it to sink.  He's been waiting for over a
>week now.  My question is:  Do all brains sink in 30% sucrose, or are the
>brains of "higher" organism too myelin-rich to sink?  Any responses will be
>appreciated.  He defends in about 3 weeks, and is a bit nervous as this work
>represents his last piece of supporting data.
Dear Karen,

I copied my answer to a similar question Marcia Bentz posed to the histonet
last year. Here it comes:

Sucrose was introduced as a cryoprotectant for cryo-ultramicrotomy  in 1973
by Tokuyasu. After chemical fixation (in your case formalin) tissue is
incubated in 50mm glycin in PBS to block residual aldehyde groups.
After this tissue is impregnated in a graded series of sucrose (
0.6M-1.0-1.5-2.3M) in 0.1M phosphate buffer. Infusion time for each
concentration of sucrose at least 1hr. For ultramicrotomy (tissue blocks
1-6mm3 ) infusion is judged to be completed if the specimens are no longer
floating on top of the sucrose solution. After cryoprotection specimens are
mounted on a stub and frozen in LN2.

Because you are using a cryostat and not a cryo-ultratome (cutting temp.
starts at -70oC) the final concentration of sucrose has to be lower. Good
results are obtained by using 5-10-20-30-40-50% sucrose in 0.1M phosphate
buffer. After infusion tissue is impregnated in OCT for approx. 15 minutes,
mounted on a stub in fresh OCT and frozen in LN2.

Detailed info on the original Tokuyasu method and alternatives can be found in:
Tokuyasu (1973) J. Cell Biol. 57, 551
Menco et al. (1978) Cell Tissue Res. 193,503
Tokuyasu (1983) J. Histochem. Cytochem. 31,164

Hope this is of help.
Bye, Peter

Peter van de Plas
Costerweg 5
6702 AA Wageningen
The Netherlands
phone: (31)-317-497676
fax: (31)-317-415955

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