Terebratalia engrailed immuno staining


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I will try using the engrailed/invected 4D9 antibody in Terebratalia.

  • Get radial, asym., bilateral, and trilobed larvae (used mixed stages from Kristineberg samples).
  • Permeabilize with 0.2% PTx (5x 5 min, 1x 30 min).
  • 2x 15 min PBT.
  • 1x 30 min 5% NGS.
  • 17h45: incubate with primary on a tube with 100 µL and dilution of 1:20.

13/10/13

  • Wash out primary antibody along the day (3x 5 min PBT, 4x 30 min PBT, 1x 30 min PTx+NGS).
  • Incubate with secondary anti-mouse-pod 1:250 overnight.

14/10/13

  • Washed 3x 5 min in PBT.
  • 4x 30 min PBT.
  • Develop with TSA and Cy5 fluorochrome (49 µL diluent + 1 µL fluorochrome).
  • Stop the reaction with 1x detergent solution at 60 °C and 2x at RT.
  • Washed 3x 5 min in 0.2% PTx.
  • Stained with phallacidin for 2h.
  • Washed 3x 5 min in PBS.
  • Grading series with TDE and mounted in 97% TDE with DAPI.

15/10/13

Confocal time. Engrailed antibody is indeed not working. I could try a higher concentration, maybe..

Ttra DAPI-Phall TDE 1.lif - Series018 Ttra DAPI-Phall TDE 1.lif - Series021 Ttra DAPI-Phall TDE 1.lif - Series024 Ttra DAPI-Phall TDE 1.lif - Series027