Lab Protocols

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 afterwar...

WHOLEMOUNT IMMUNO Fix tadpoles overnight @ 4 ° C in 4% Paraformaldehyde in 0.1M P.B. (aliquots in freezer) Rinse in 0.1M Phosphate Buffer Dissect out brains Pre-incubate in blocking solution in cold room on shaker or at room temperature for ~ 30 min Drain and replace with 1 ° antibody solution Incubate for 24hours in cold room on shaker Wash 3 x 30 min with blocking solution Wash overnight in blocking solution (4 th wash…) Drain and add 2 ° antibody solution Incubate overnight in cold room on shaker Rinse 3 x 30 min in phosphate buffer wash overnight in phosphate buffer (4 th wash…) Place brains on slide (Opening of 4 th ventricle indicates dorsal side) Mount in DAKO cytomation mounting medium using insect pins as spacers Seal slides with nail polish – we didn’t quite do this! Recipes: Blocking Solution 0.3 % triton-x-100 5 % Normal Goat Serum (or goat seru...

  4% Paraformaldehyde in 0.1M Phosphate Buffer. (Use special glassware for fix.)  To make 500mL: 1) Heat milli-Q water (200mL) to about 80 C using water bath.  In hood: 2) Place hot water from step 1 on a stir plate. 3) Add 20 g paraformaldehyde crystals while stirring. 4) Add drops (about 20) of 1.0N NaOH slowly until paraformaldehyde is well dissolved. 5)   Add 250mL 0.2M sodium phosphate buffer      (210mL dibasic and 40mL monobasic). 6)   Transfer to graduated cylinder and add milli-Q water to get a final volume of 500mL. 7)   Test pH of a small aliquot using pH test paper.   Adjust to approx pH 7.4. 8)   Filter and store in a fixative fridge.

Prepare the following solutions: (To determine the amount needed for your experiment, please note that one tube of Solution 1 will yield a total of 100mL of working solution, at a final concentration of 0.5ug/mL.) Solution 1*: 5 μL DAPI (10 mg/ml solution) 995 μL PB * Solution is viable for one month if stored at 4° in the dark Solution 2 (working solution): 10 μL of Solution 1 990 μL PB Treatment: →Add Solution 2 (working solution) to your tissue/cells and incubate at room temperature for 1- 5 minutes. →Rinse 2X with PB. →Mount coverslip with Shur/Mount, Prolong or comparable antifade medium. →Seal coverslips (if necessary) with Valap or nail polish.** **Try to avoid nail polish if are visualizing GFP, as the additives in nail polish tend to quench the signal.

1. Incubate 2 x 20 min. in 0.1M PB at R.T. 2. Incubate 4 x 10 min in 0.1M PB + 0.3% triton at R.T. 3. Incubate 30 min in 0.1M PB + 0.3% triton +5% NGS at RT 4. Incubate overnight with primary antibody _______________ Diluted _____________ in 0.1M PB + 0.03% triton at 40C. 5. Rinse 4 x 10 min in 0.1M PB + 0.3% triton at RT 6. Incubate slides 1 hr in secondary antibody _______________ Diluted _____________ in 0.1M PB + 0.03% triton at R.T. 7. Rinse 3 x. 10 min in 0.1M PB at R.T. 8. 3 drops mounting medium + coverslip. Store at –200C