Fix tissue quickly by dipping small slices (less than 1 mm-thick in at least one direction) into 2.5% gluteraldehyde and 2% paraformaldehyde in 0.1M sodium cacodylate buffer (pH 7.4). Chop into small 1 mm3 cubes in the hood and leave to fix for 1 hour @ RT.
Wash with 0.1M sodium cacodylate buffer 3 x 20 minutes.
Post Fixation
Postfix in 1% osmium tetroxide in 0.1M cacodylate buffer for 1 hour @ RT in the hood.
En Bloc Staining
Wash in 50mM sodium maleate buffer (pH 5.2) 3 x 15 minutes.
Stain in 2% uranyl acetate in maleate buffer for 1 hour @ RT in the dark.
Dehydration
Wash in water 3 x 5 minutes.
Dehydrate cells in the following order:
50% ethanol 2 x 5 minutes
70% ethanol 2 x 5 minutes
90% ethanol 2 x 5 minutes
100% ethanol 3 x 10 minutes
Replace ethanol with propylene oxide
Leave in fresh propylene oxide for 10 minutes with the lid closed.
Infiltration
Replace with 50% propylene oxide / 50% Epon mix. Leave on the wheel for 2 hours with the lid closed.
Replace with pure Epon and leave on the wheel for 2 hours with lid open. Repeat once.
Transfer samples to fresh Epon in moulds or Beem capsules (remove air bubbles). Add computer-printed labels. Cure in the oven overnight @ 60°C.
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