Terebratalia segmentation in situ

In order to have more temporal resolution of engrailed I am adding one well per stage. In addition some segmentation related genes.

en cleavage+blastula en radial gastrula en asym gastrula en bilateral gastrula en trilobed+early+late larva
pax6 nk1a nk1b lmx1
 lfng runx hh

 25/11/14

Washed probe out. Added antiDIG-AP antibody at 1700 and incubated overnight at 4 °C.

26/11/14

Washed antibody and started developing. Pax6 came up quite fast. I exchanged the solution after 1h and stopped after 2h. The remaining are slowly coming up, except asym and bilateral of engrailed. I used one tube of 0.8 ng/µL of probe and it must have been even lower concentration because the other 3 engrailed wells are coming up ok (same probe batch, but from another tube). Another issue is that there is not early gastrula like I wanted, they seem to be already have the differentiated lateral patches.

Lunatic fringe and runx are not showing up. I exchanged AP from all wells except engrailed after 3h and then all wells before leaving at 4 °C overnight.

27/11/14

Same as yesterday with NK1s and lmx coming up nicely. Exchanged AP at 10:30.

28/11/14

Kept developing for 7h and stopped all wells. Nothing came up for lfng, runx or hedgehog.

30/11/14

Ethanol washes for all. Added 70% glycerol with 1:10000 sytox green in hedgehog well. I want to see if embryos turn red… If not I’ll check if it is better than DAPI for these regular in situs.