Novocrania and double in situs of Terebratalia

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.