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