Began in situ 10 min protk (half-concentrated for Nano) with:
Ttra wnt2 |
Ttra wnta |
|
Nano wnt8 |
Nano wnt11 |
Nano pax2/5/8 |
07/12/13
Added probes at 13:00.
09/12/13
First day of washes, added anti-dig-ap around 18h.
10/12/13
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.
11/12/13
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.
12/12/13
Ethanol washes, then first wash with glycerol and let in nutator for 3h. Exchange glycerol for glycerol+dapi and let it overnight.
13/12/13
Mounted slides of Novocrania and took pictures.