Fixation of Xenopus tadpoles for cryosectioning
Fixation of Xenopus tadpoles for cryosectioning
Fix tadpoles in 4%PFA/0.1M PB for 1h at room temperature on the shaker
Transfer tadpoles to cold Dent’s Fixative (80% Methanol/ 20% DMSO, store at -20°C)
Fix overnight at -20°C (at this step tadpoles can be stored for a longer time at -20°C)
Wash tadpoles in 100mM Tris/HCl pH 7.4, 100mM NaCl for 1h at room temperature on the shaker
Transfer tadpoles to 15% Fish Gelatin/ 15% Sucrose, incubate o/n at room temperature on the shaker
Transfer tadpoles to 25% Fish Gelatin/ 15% Sucrose, incubate o/n at room temperature on the shaker
Embed tadpoles in 20% Fish Gelatin/ 15% Sucrose for cryosectioning
Fish Gelatin, from Norland (HiPure Liquid Fish Gelatin HP-03)
Attention when preparing the solutions since the fish gelatin stock solution is just a 45% solution
Don’t measure fish gelatin with cylinder but weigh it on the balance
Estimate volume of fish gelatin by eye
Dissolve sucrose in water then add to fish gelatin afterwards, not possible to dissolve sucrose in fish gelatin
Mix sucrose and fish gelatin really well, need to stir for a while
For long term storage keep at 4°C
Fix tadpoles in 4%PFA/0.1M PB for 1h at room temperature on the shaker
Transfer tadpoles to cold Dent’s Fixative (80% Methanol/ 20% DMSO, store at -20°C)
Fix overnight at -20°C (at this step tadpoles can be stored for a longer time at -20°C)
Wash tadpoles in 100mM Tris/HCl pH 7.4, 100mM NaCl for 1h at room temperature on the shaker
Transfer tadpoles to 15% Fish Gelatin/ 15% Sucrose, incubate o/n at room temperature on the shaker
Transfer tadpoles to 25% Fish Gelatin/ 15% Sucrose, incubate o/n at room temperature on the shaker
Embed tadpoles in 20% Fish Gelatin/ 15% Sucrose for cryosectioning
Fish Gelatin, from Norland (HiPure Liquid Fish Gelatin HP-03)
Attention when preparing the solutions since the fish gelatin stock solution is just a 45% solution
Don’t measure fish gelatin with cylinder but weigh it on the balance
Estimate volume of fish gelatin by eye
Dissolve sucrose in water then add to fish gelatin afterwards, not possible to dissolve sucrose in fish gelatin
Mix sucrose and fish gelatin really well, need to stir for a while
For long term storage keep at 4°C
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