Wholemount immuno standard

WHOLEMOUNT IMMUNO

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 (4^th 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 (4^th wash…)
13. Place brains on slide (Opening of 4^th 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!

Recipes:

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.