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.


Added probes at 11h.


Washing day embryos looking good.


Washed out antibody with long 30 min washes.


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.


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


Restarted the wnts to see if something comes up…


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


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