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 (4th
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 (4th wash…)
- Place
brains on slide (Opening of 4th 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 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.