IV.Whole mount immunostaining

Method
1. Collect embryos in ice cold PBS.
2. Fix the embryos in freshly prepared 4% paraformaldehyde in PBS 1.5 hr.
3. Wash the embryos in PBS , three times for 15 min.
4. Incubate the embryos overnight with freshly made bleaching solution.
5. Wash the embryos in 1% Tx-100, 10% fetal calf serum in PBS (TS-PBS), three times for lhr.
6. Incubate embryos with primary antibody (mouse monoclonal antibody against the 155 x 103Mr neurofilament protein: 2H3 supernatant diluted 1:1 in TS-PBS) for 3 days.
7. Wash embryos with TS-PBS 6 times for 30 min.
8. Incubate with peroxidase-conjugated goat anti-mouse immunoglobulins (diluted 1/200 in TS-PBS) overnight.
9. Wash embryos with PBT 6 times for 30 nun.
10. Bring embryos to room temperature 5 min. before colour development.
11. Incubate embryos in freshly prepared solution A for 15 min., solution B three times for 10 min., and solution C for 3-10 min. at room temperature Monitor colour development of embryos in solution C. Stop reaction by washing in 30% ethanol three times for 1 hr., then 16 hr.
12. Clear embryos with glycerol for photography.

*All incubation and washing steps are carried out at 40C with gentle rocking, except for colour development steps.

Materials
1 1OXPBS
2 4% Paraformaldehyde in PBS
3 Bleaching solution: 0.05% H202, 0.2% Tx-l00 in PBS
4 Blocking solution (TS-PBS): 1% Tx-100, 10% fetal calf serum in PBS
5 Washing solution (PBT): 0.2% Tx-100 in PBS
6 primary Ab - 1/2 dilution with TS-PBS
7 secondry Ab - 1/200 dilution with TS-PBS
8 Solution A :10 mg 4-chloro-1 naphtol + 100 ul 100% Ethanol, stir in 25 mi 0.1 M Tris pH 7.6 and 0.1% Triton X-l00, filter
9 Solution B:10 mg 4-chloro-1 naphtol + 100 ul 100% Ethanol, stir in 25 mi 0.1M Tris pH
7.6, filter
10 Solution C : 25 mg 4-chloro-1 naphtol + 4 mi Ethanol, + 6 mi H20, then 2u' H202 right before use.