Brachiopod mesoderm immunostaining

Mesoderm and maybe some neurons and muscles. Engrailed antibody second try
Ttra mixed stages: DAPI / PHALL / Tyros. Tub. 1:500 a594 (mouse) / Synapsin II 1:500 a647 (rabbit) Ttra mixed stages DAPI / PHALL / Serotonin 1:500 a647 (rabbit) / Engrailed 4D9 1:25 a594 (mouse)
Nano mixed stages: DAPI / PHALL / Tyros. Tub. 1:500 a594 (mouse) / Synapsin II 1:500 a647 (rabbit) Nano mixed stages DAPI / PHALL / Serotonin 1:500 a647 (rabbit) / Engrailed 4D9 1:25 a594 (mouse)
  • 2h permeabilizing in 0.2 % PTx
  • 2h 0.2 % PTx + BSA.
  • 1h 0.2 % PTx + 5 % NGS.
  • Incubated with primary antibodies at 4°C in 0.2% PTx+NGS.

14/02/15

  • Washed PTx+BSA 3x 5 min.
  • Washed PTx+BSA 4x 30 min.
  • Incubated 1h in PTx+NGS.
  • Added secondaries (alexa 594 mouse and alexa 647 rabbit) in a concentration of 1:200.

15/02/15

  • Washed PTx+BSA 3x 5min.
  • Washed PBT 5x 10 min.
  • Stained with BODIPY FL and DAPI (1:500) for 2h.
  • Washed out staining with 1x PBS.
  • Overnight at 4 °C.

16/02/15

Mounting with Murray Clear went well.