Novocrania and double in situs of Terebratalia
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| Nano ptc1 | Nano smo1 | Nano smo2 | Nano gli |
| Nano pax6 | Nano delta | Nano notch | |
| Ttra pax6 (DNP/cy5) + pax258 (DIG) | Ttra en (DNP) + pax6 (DIG) | Ttra wnt1 (DNP) + delta (DIG) | Ttra wnt1 (DNP) + pou4 (DIG) |
28/11/14
Added probes at 1 ng/µL concentration.
30/11/14
Washes, incubated with the blocking for 1h and at 4 °C overnight with the respective antibodies. Anti-DIG-AP 1:5000 for all Novocrania wells and Anti-DIG-POD 1:250 for all Terebratalia wells.
01/12/14
Washed antibody from all samples with:
- 5x 15 min PBT and 5x 30 min PTw.
- 3x 5min washes of AP minus MgCl2 for regular in situ and TNT buffer for fluorescent in situs.
Developed fluorescent wells with TSA kit using cy3 fluorochrome for 2 hours. Sropped the reaction with 5x 5 min detergent solution at 60°C followed by PTw washes. POD inactivation with 0.1% H2O2 for 45 min, PTw washes, and finally formamide based POD inactivation buffer for 15 min at 60 °C. Signal for pax6 and delta were very strong, pou4 was strong and pax258 was medium.
I started developing Novocrania with NBT/BCIP. Pax6 was the first to come, strongly. The others were slower, but gli had a good signal/noise ratio. Others acquired background after the first AP change after 2 hours. I stopped all, except delta and smo2 (which had less background).
02/12/14
Today they were quite dark :/
Went through ethanol washes and antibody washes for fluorescent. Added 70% glycerol in PTw with 1:10000 sytox green.
