2019-08-06T00:18:56Z (GMT) by
The mouse was anesthetized with Nembutal and then perfused with Ringer's containing heparin and xylocaine and bubbled with 95% O2 / 5% CO2. The mouse was then perfused with 2.5% glutaraldehyde / 2% PFA in cacodylate buffer containing 2 mM CaCl.The brain was removed and placed in same post-fix for 2 hours and 15 minutes in fridge.The brain was cut in to 100 um thick vibratome sections in ice cold cac buffer with 2mM Ca.The slices were washed in cac w/ Ca for 30 minutes in fridge.The slices were placed into 2% OsO4 / 1.5% potassium ferrocyanide in cac w/ Ca at room temp for 1 hour.The slices were washed 3x 2 minutes with ddH2O and then placed into 1% aq. thiocarbohydrazide for 20 minutes at room temp.The slices were washed 2x 2 minutes with ddH2O and then placed in 2% aq. OsO4 for 30 min at room temp.The slices were washed 3x 2 min with ddH2O and then placed in 2% aq. UA overnight in fridge.The next day the slices were washed 3x 10 min in ddH2O and then placed in lead aspartate solution in 60 degree oven for 30 min.The slices were washed 3x 5min in ddH2O at room temp.The slices were dehydrated in series of ice cold EtOH solutions (70%, 90%, 100%, 100%; 10 min each).The slices were placed in ice-cold acetone for 10 min and left at room tempThe slices were placed in room temp acetone for 10 minutes.The slices were placed in 25% Durcupan for 3 hours. The slices were placed in 50% Durcupan overnight.The slices were placed in 75% Durcupan for 3 hours. The slices were placed in 100% Durcupan overnight.The slices were flat-embedded in fresh 100% Durcupan and left in 50 degree oven for 48 hours.