Terebratalia Azakenpaullone treated in situ wnt1 and engrailed

Starting in situ with azakenpaullone-treated Terebratalia embryos with wnt1.

Blastula Mid Blastula Mid blastula to larva Early Gastrula Early Larva
1 µM 1 µM 1 µM 1 µM 1 µM
10 µM 10 µM 10 µM 10 µM 10 µM
C- C- C- C- C-

Prepared fresh PTw and went up to hybe step. Then split samples for wnt1 and engrailed (30 wells in total). Wnt1 probe will be fine, but engrailed will be much less concentrated, probably around 0.1 ng/µL. Unfortunately this is what I had in stock.

06/04/14

I fell face to the ground and could not go to the lab. Chema put my 2 plates in the freezer.

07/04/14

I put the plates back at 62 °C for 5h. I diluted wnt1 probes and added the usual 500 µL volume to the wells. For the engrailed probe I had to dilute it to 0.8 ng/µL and use only 250 µL of volume… but well.

09/04/14

Washing day. Everything went normally. I added the antibody around 17:30.

10/04/14

Washed 2 x 15 min and 1x 1h with PBT and then 1x 15 min. Finally 5x 30 min with PTw. Left the plates in the cold room.

11/04/14

Developing day. Ran for 5h and some signal is appearing; stronger in the treated embryos than in the controls. Exchanged the AP substrate at 17:30 and put in the cold room.

12/04/14

Most of the treated embryos have a nice signal. Controls are surprisingly faint, though. I developed through the whole day and kept in the cold room overnight. Probably stopping tomorrow some wells.

13/04/14

Stopped the reactions in the treated samples with 3 washes of AP without magnesium chloride. I’m still developing the controls.

14/04/14

Still developing controls. Stopped wnt1, but not engrailed.

15/04/14

Exchanged again the AP. It looks much better now, signal is stronger. I did not filter the AP this time to see if I get crystals.

Developed for 8h and stopped the reaction on the last wells.

16/04/14

Ethanol washes upt to glycerol.