Since mounting with TDE was not so good (crystals and some weird noisy background) I am staining with propidium iodide and phallacidin and mounting with murray clear. This should give a clean embryo with the mesodermal signal visible enough for details. Fluorochrome for engrailed is Cy5.
Confocal.
Began in situ 10 min protk (half-concentrated for Nano) with:
| Ttra wnt2 | Ttra wnta | |
| Nano wnt8 | Nano wnt11 | Nano pax2/5/8 |
Added probes at 13:00.
First day of washes, added anti-dig-ap around 18h.
Washing off antibody, starting with 2x 15min PBT and after 1h 3x 15min PBT and normal PTw washes.
Started developing at 16h. Some signal started to appear at Wnt11 ~18h30.
Arrived 9h30 and the wells were purple! Did an extra wash with AP substrate and let at RT. Exchanged AP 4 times and stopped the reaction at 19h with 3x AP without MgCl2 and 5x PTw. Kept at 4 °C overnight.
Ethanol washes, then first wash with glycerol and let in nutator for 3h. Exchange glycerol for glycerol+dapi and let it overnight.
Mounted slides of Novocrania and took pictures.
Probe PCR with 6 samples:
Set transcription reaction for all except for MP68 at 10h30. Began precipitation with 5 µL of lithium.
| ng/µL | |
| Ttra wnt2 | 1008 |
| Ttra wnta | 801 |
| Nano wnt8 | 2000 |
| Nano wnt11 | 1353 |
| Nano pax258 | 860 |