1. Fix tadpoles overnight @ 4°C in 4% Paraformaldehyde in 0.1M P.B. (aliquots in freezer)
  2. Rinse in 0.1M Phosphate Buffer
  3. Dissect out brains
  4. Pre-incubate in blocking solution in cold room on shaker or at room temperature for ~ 30 min
  5. Drain and replace with 1° antibody solution
  6. Incubate for 24hours in cold room on shaker
  7. Wash 3 x 30 min with blocking solution
  8. Wash overnight in blocking solution (4th wash…)
  9. Drain and add 2° antibody solution
  10. Incubate overnight in cold room on shaker
  11. Rinse 3 x 30 min in phosphate buffer
  12. wash overnight in phosphate buffer (4th wash…)
  13. Place brains on slide (Opening of 4th ventricle indicates dorsal side)
  14. Mount in DAKO cytomation mounting medium using insect pins as spacers
  15. Seal slides with nail polish – we didn’t quite do this!





Blocking Solution

0.3 % triton-x-100

5 % Normal Goat Serum (or goat serum)

in 0.1 M Phosphate buffer


1° antibody solution

1:1000 (antibody : blocking solution) works adequately with anti-myc (Invitrogen)

1:5000 – labeled cell bodies well but not axons.


2° antibody solution

1:100 (antibody : blocking solution)   


the volume in each well, depending on the dish used, can vary but at 100 uL you get good immuno staining.  The washes can be done at ~ 200 uL to get a thorough cleansing.