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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.

实验小鼠经 Nembutal 麻醉后，采用含有肝素和罗哌卡因的 Ringer 溶液进行灌注，并使用 95% 氧气 / 5% 二氧化碳气泡处理。随后，小鼠被灌注以 2.5% glutaraldehyde / 2% PFA 的 cacodylate 缓冲液，其中含有 2 mM 的 CaCl2。随后取出脑部，并将其置于相同的固定液中在冰箱中保存 2 小时 15 分钟。脑部经振动切片机切割成 100 微米厚的切片，并在含有 2mM Ca 的冰冷 cac 缓冲液中处理。切片在冰箱中的 cac w/ Ca 缓冲液中清洗 30 分钟。随后，将切片置于室温下 2% OsO4 / 1.5% 钾铁氰化物溶液中的 cac w/ Ca 缓冲液中 1 小时。切片用 ddH2O 清洗 3 次，每次 2 分钟，然后置于室温下 1% 水溶性硫代碳酰胺溶液中 20 分钟。切片再次用 ddH2O 清洗 2 次，每次 2 分钟，然后置于室温下 2% 水溶性 OsO4 中 30 分钟。切片用 ddH2O 清洗 3 次，每次 2 分钟，随后置于冰箱中 2% 水溶性 UA 溶液中过夜。次日，切片用 ddH2O 清洗 3 次，每次 10 分钟，然后置于 60 度的铅天冬氨酸溶液中 30 分钟。切片在室温下用 ddH2O 清洗 3 次，每次 5 分钟。切片依次在冰冷的乙醇溶液（70%、90%、100%、100%；每次 10 分钟）中进行脱水处理。切片在冰冷的丙酮中处理 10 分钟，并在室温下放置。切片在室温下的丙酮中处理 10 分钟。切片置于 25% Durcupan 溶液中 3 小时。切片在 50% Durcupan 溶液中过夜。切片在 75% Durcupan 溶液中处理 3 小时。切片在 100% Durcupan 溶液中过夜。切片在新鲜配制的 100% Durcupan 溶液中进行平铺包埋，并在 50 度的烤箱中放置 48 小时。