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Tissue group type
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Astrocyte Soma Reconstruction
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| Protocol used |
Perfuse animal with Ringer's solution for 2 minutes followed by 2% glutaraldehyde and 2% paraformaldehyde in 0.15M sodium cacodylate buffer, pH 7.4, at 35 degrees Celsius for 10 minutes. Remove tissue and fix on ice for 1-2 hours. During this time the tissue is further dissected.
Wash 5x5 minutes in cold 0.15M sodium cacodylate buffer.
Postfix in 1% OsO4 in 0.15M sodium cacodylate buffer on ice for 1 hour.
Rinse in cold ddH2O 4x5 minutes.
Place tissue into 2% uranyl acetate in ddH2O on ice for 2 hours.
Rinse tissue in ice cold ddH2O 3x5 minutes.
Dehydrate tissue in ethanol series 20, 50, 70, 90, 100% for 5 minutes each on ice.
Complete dehydration with 100% acetone 2x10 minutes, first on ice, second at room temperature.
Infiltrate with solution of 50% acetone and 50% durcupan ACM resin overnight in scintillation vials with caps on.
Transfer tissue to foil dishes and change to 100% resin for 1 hour. Place into fresh resin for an additional 2 hours.
Place in fresh resin and flat embed using 2 mould release treated slides. Polymerize in 60 degree Celsius vacuum oven for 24-48 hours.
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