|Affiliation||Department of Cell Biology, Graduate School of Medicine, Osaka University|
|Contact||inquiry about this protocol|
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- fixation by perfusion through the heart with 1/2 Karnovsky fixative
- take out the sample and wash with 10% sucrose in 0.1M cacodylate buffer. 5min on ice
- fix with 1% osmic acid 1hr on ice (DO NOT fix more than 1.5hrs) * 1% osmic acid: 2% osmic acid in water ( ampule bought from TAAB Co.):0.2M cacodylate buffer=1:1
- wash with D.W. three times *aspirate DW using an aspirator in the draft chamber
- block stain with 0.5% uranil acetate in D.W 2hrs- (O/N) * If you want to dehydrate during that day, you can use 1% uranil acetate in D.W, 1hr and then dehydrate
- dehydration starting from 65%EtOH→75%EtOH→85%EtOH→95%EtOH→99%EtOH 5min each
- dehydrate in 100%EtOH, 20min 3times (total 60min)
- switch to proprene oxide, 20min twice
- pour mixture of Epon:propyrene oxide=1:1 to alminium dish *make the mixture beforehand with 50ml Falcon plastic tube
- transfer the samples from the vial to the alminium dish with tweezers and leave the alminium dish in the dissicator * wait at least overnight to evaporate propyrene oxide if it is inadequate, leave them in the dissicator for another night
- change the mixture with Epon and remove air by a pump( to remove proprene oxide completely) *pour epon into the alminium dish. Pick up samples in the mixture of epon and propyrene oxide take of the mixture as much as possible by rolling the sample on the ‘Kim Wipe’. After removing the mixture, put the samples into the alminium dish filled with epon. Put the samples in the alminium dish into the dissicator and remove air by pumping for about four hours.
- change epon and remove air by pumping, O/N
- take the samples (in the alminium dish) out of the dissicator and put them into the oven which is kept 65Co and polymerize the epon more than 36hrs ※Garbage with Epon (e.g., alminium dishes, plastic tubes, etc.) should be put in the oven which is kept at 50Co before discarding