Novocrania in situ 3rd try 


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Third try with engrailed, wnt1, and wnt5. This time using half-concentrated 5 min ProteinaseK treatment. They should not get damaged!

  • Fixed for 1h25min.
  • Washed normally and incubated in pre-hybe.

28/10/13

Added probes at 11h.

30/10/13

Washing day embryos looking good.

31/10/13

Washed out antibody with long 30 min washes.

01/11/13

Started developing. Engrailed began to appear after 2hs, but nothing in the wnts. I exchanged the AP substrate every 2 hours. At 18h I transferred to new wells because crystals were forming. At 19h30 I exchanged the AP and put them in the cold room.

02/11/13

Stopped engrailed and continued developing the wnts during the day. No signal came up except for background. Paused in AP without magnesium.

04/11/13

Restarted the wnts to see if something comes up…

05/11/13

Stopped wnts… Ethanol washes and added glycerol+DAPI overnight in cold room.

06/11/13

Mount slides. Engrailed has some shitty background… Remember next time to stop before overnight.